Ciliary Neurotrophic Factor Receptor Ligand-Binding Agents and Methods of Using the Same

ABSTRACT

Provided are agents that specifically bind a ligand of ciliary neurotrophic factor receptor (CNTFR). In certain aspects, an agent of the present disclosure is a soluble CNTFR polypeptide. The soluble CNTFR polypeptide may have an altered (e.g., reduced) binding affinity for one or more ligand-CNTFR complex subunits, an altered (e.g., increased) binding affinity for one or more CNTFR ligands, or any combination thereof. Compositions that include the agents of the present disclosure are also provided, as are methods of using the agents (e.g., for treating a cell proliferative disorder) and methods of identifying an individual as having a cell proliferative disorder associated with CNTFR signaling.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/430,757, filed Dec. 6, 2016, and U.S. ProvisionalPatent Application No. 62/481,027, filed Apr. 3, 2017, whichapplications are incorporated herein by reference in their entirety.

INTRODUCTION

Ciliary neurotrophic factor (CNTF) was identified as a survival factorfor chick ciliary neurons and belongs to the interleukin (IL)-6 familyof structurally related hemato- and neuropoietic cytokines (IL-6, IL-11,cardiotrophin-like cytokine factor 1 (CLCF1), leukemia inhibitory factor(LIF), oncostatin M (OSM), cardiotrophin-1 (CT-1)). Cellular responsesto CNTF and IL-6 type cytokines are elicited by different multi-unitreceptor complexes that include the membrane-spanning 130-kDaglycoprotein, gp130. CNTF first binds in a 1:1 stoichiometry to theGPI-anchored CNTF receptor (CNTFR), which is not involved in signaltransduction. Binding of CNTF to the membrane-bound or soluble CNTFRinduces a heterodimer of the signal transducing β-receptors gp130 andLIF receptor (LIFR), which triggers intracellular signaling cascades.

Cancer is initiated and progresses within a microenvironment that isitself altered as a consequence of the tumorigenic process. Stromalcells in contact with cancer cells secrete growth factors and cytokinesthat may act directly by signaling to tumor cells or indirectly byrecruiting other stromal components to promote tumor progression. Animportant aspect of this process is the expansion of cancer-associatedfibroblasts (CAFs). CAFs are a diverse population of stromal cells withdistinct characteristics in different tumors and tissues.

CAFs support the growth of cancer cells (e.g., lung cancer cells) invivo by secretion of soluble factors that stimulate the growth of tumorcells. One such soluble factor is cardiotrophin-like cytokine factor 1(CLCF1). CLCF1 produced by cells in the stroma is received as a growthsignal by tumor cells expressing a receptor for this protein—CNTFR. Forexample, functional studies have identified a role for CLCF1-CNTFRsignaling in promoting growth of non-small cell lung cancer (NSCLC).

SUMMARY

Provided are agents that specifically bind a ligand of ciliaryneurotrophic factor receptor (CNTFR). In certain aspects, an agent ofthe present disclosure is a soluble CNTFR polypeptide. The soluble CNTFRpolypeptide may have an altered (e.g., reduced) binding affinity for oneor more ligand-CNTFR complex subunits, an altered (e.g., increased)binding affinity for one or more CNTFR ligands, or any combinationthereof. Compositions that include the agents of the present disclosureare also provided, as are methods of using the agents (e.g., fortreating a cell proliferative disorder) and methods of identifying anindividual as having a cell proliferative disorder associated with CNTFRsignaling.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates a strategy to inhibit downstream CNTFRsignaling pathways according to one embodiment of the presentdisclosure. In this embodiment, the agent that specifically binds aligand of CNTFR is a soluble CNTFR “decoy receptor” that inhibits CLCF1from interacting with membrane bound CNTFR, preventing activation ofCLCF1-CNTFR mediated signaling pathways such as the JAK STAT pathway andthe MAPK/ERK pathway.

FIG. 2 provides data demonstrating that recombinant CLCF1 activatesSTAT3. Panel A: The level of p-STAT3 at Tyr705 in the NSCLC cell lineA549 was detected 15 min and 30 min after treatment. Panel B: Intensityof p-STAT3 signal was quantified after normalized by CNTFR expression.Panel C: STAT3 activation was detected in other NSCLC cell lines such asH23 and H358.

FIG. 3 shows data demonstrating the effect of CLCF1 treatment on A549and H23 cell survival. After 24 hours of starvation in serum-freeconditions, cells were treated with CLCF1 in serum free media for 72 h.CLCF1 increased A549 (panel A) and H23 (panel B) cell survival in aconcentration dependent manner.

FIG. 4 Yeast surface display of CNTFR. Panel A: CNTFR was displayed as afusion to yeast surface protein Aga2p. The displayed protein contains ac-terminal c-myc tag, which allows expression levels to be measured.Panel B: Flow cytometry scatter plot showing that when treated with 20nM CLCF1, the yeast population expressing CNTFR has increased signal forCLCF1 binding.

FIGS. 5A-5E Yeast-displayed CNTFR binds to the beta receptors in thepresence of CLCF1. Scatter plots showing CNTFR displaying yeastincubated with anti-cMyc antibody only (panel A), with LIFR-Fc (panelB), with CLCF1 and LIFR-Fc (panel C), and with CLCF1, LIFR-Fc, andgp130-HIS (panel D). Fluorescently labeled secondary antibodies wereused to detect binding of the various components by flow cytometry.Panel E: Overlay of the plots from panels B, C, and D.

FIGS. 6A-6E Yeast-displayed CNTFR-CLCF1 can bind to gp130 without LIFR.Scatter plots showing CNTFR displaying yeast incubated with anti-cMycantibody only (panel A), with gp130-Fc (panel B), with CLCF1 andgp130-Fc (panel C), and with CLCF1, gp130-Fc, and LIFR-HIS (panel D).Fluorescently labeled secondary antibodies were used to detect bindingof the various components by flow cytometry. Panel E: Overlay of theplots from panels B, C, and D.

FIG. 7 shows variants isolated from the intermediate affinity populationafter 3 rounds of sorting of the randomly mutagenized CNTFR libraryagainst CLCF1.

FIG. 8 shows variants isolated from the highest affinity populationafter 3 rounds of sorting of the randomly mutagenized CNTFR libraryagainst CLCF1.

FIG. 9 shows variants isolated from the highest affinity populationafter 3 rounds of sorting of the shuffled CNTFR library against CLCF1.

FIGS. 10A-10D characterization of affinity matured CNTFR variants. PanelA: CLCF1 binding to yeast expressing CNTFR constructs isolated fromaffinity maturation screens. Panel B: Apparent Kd values of yeastdisplayed CNTFR constructs. Panel C: CNTF binding to yeast displayedwild-type CNTFR and variant 4. Panel D: Variant 4 bound to mCLCF1 inaddition to hCLCF1.

FIG. 11 A CNTFR variant containing T268A and D269A mutations binds toCLCF1 and LIFR, but not gp130. CNTFR T268A D269A construct bindingsignals to CLCF1 (panel A), gp130-Fc (panel B), and LIFR-Fc (panel C).

FIGS. 12A-12D Library screening for CNTFR variants that do not bind toLIFR. After the initial screening was carried out for high affinityCLCF1 binders, a population with low LIFR-Fc signal was isolated (panelA). This was followed by another screening for CLCF1 binders (panel C)and a screening for non-LIFR binders. Panel D: After 6 rounds of sortingthe screened library shows decreased LIFR binding while it had unchangedCLCF1 binding. Gates in A upper panel, B lower panel, and C lower panelrepresent the isolated population at each sort round.

FIG. 13 Characterization of a CNTFR variant that does not bind LIFR.Panel A: The variants isolated from the library screening approach ofusing negative sorts against LIFR (neg. sort), had two consensusmutations, Y177H and K178N. Panel B: These two mutations shows additiveeffects on decreasing LIFR binding affinity when combined. V4=variant 4from FIG. 10.

FIG. 14 Characterization of soluble eCNTFR constructs. Panel A: Solublyexpressed eCNTFR-HIS and eCNTFR-Fc showed significantly improved bindingto CLCF1 compared to wtCNTFR proteins. Panel B: Soluble eCNTFRconstructs showed minimal binding to soluble gp130 and LIFR (for eachcondition, from left to right: wtCNTFR-HIS; wtCNTFR-Fc; eCNTFR-HIS;eCNTFR-Fc).

FIGS. 15A-15C Effects of eCNTFR-Fc on cell signaling and survival. PanelA: A549 non-small cell lung cancer cells were treated with 10 nMCNTFR-Fc variants for 20 minutes and western blot was performed todetect pSTAT3. eCNTFR-Fc treatment inhibited the survival-enhancingeffect of CLCF1 in A549 (panel B) and H23 cells (panel C). For panels Band C, for each concentration, results for CLCF1 are on the left andthose for CLCF1+eCNTFR-Fc are on the right.

FIGS. 16A-16C Sequestration of CLCF1 by eCNTFR-Fc inhibits tumor growthin an A549 xenograft model. Panel A: CLCF1 sequestration and clearanceof eCNTFR-Fc throughout 48 hours after 1 mg/kg body weight dose. PanelB: Tumor burden in xenograft model of A549 NSCLC cells. Mice weretreated with PBS, 1 mg/kg body weight, or 10 mg/kg body weight eCNTFR-Fctwice weekly (arrow) for 17 days (circles: PBS, squares: 1 mg/kgeCNTFR-Fc, triangles: 10 mg/kg eCNTFR-Fc). Panel C: Waterfall plot ofthe individual tumor sizes normalized from time 0.

FIG. 17 eCNTFR-Fc inhibits tumor growth in the H23 xenograft model.Panel A: Tumor burden in xenograft model of H23 NSCLC cells. Mice weretreated three times per week using PBS (circles), 10 mg/kg body weightwtCNTFR-Fc (squares) or eCNTFR-Fc (triangles) over 39 days. Panel B:Waterfall plot of the individual tumors.

FIG. 18, panels A and B, shows data demonstrating increased specificityand in vitro efficacy in engineered CLCF1 trap (eCNTFR). Shown iseCNTFR-Fc+mCLCF1 (circles); wtCNTFR-Fc+CNTF (triangles);wtCNTFR-Fc+mCLCF1(diamonds); and eCNTFR-Fc+CNTF. Inhibition of STAT3phosphorylation was used to demonstrate inhibitory effect.

FIGS. 19A-191, provide data showing the in vivo effect of eCNTFR. PanelA: Blood clearance and CLCF1 sequestration after intraperitoneal (i.p.)dosing of 10 mg/kg eCNTFR-Fc in NOD/SCID/gamma mice. Serum samples werecollected post injection and unbound CLCF1 was measured by ELISA usingeCNTFR-Fc as the capture agent. Vehicle-treated mice were used todetermine baseline CLCF1 levels. eCNTFR-Fc levels in the blood werequantified by ELISA using an anti-Fc antibody as the capture agent.Panel B: Tumor volume quantification of A549 xenografts [n=8 tumorsexcept PBS (n=6 tumors at the last time point), one experiment]. *P<0.05; ** P<0.01; *** P<0.001 using two-way ANOVA. Data are representedas mean±s.e.m. Panel C: Tumor volume quantification of final time pointA549 xenografts final time point. Whiskers identify the maximum andminimum values; boxes indicate the 75^(th) and 25^(th) percentile andthe line the median. * P<0.05; ** P<0.01 using one-way ANOVA. Panel D:Waterfall plot showing tumor percent change from baseline for A549xenografts. Panel E: Tumor volume quantification of patient-derivedxenograft (PDTX) model. ** P<0.01; **** P<0.0001 using two-way ANOVA.Data are represented as mean±s.e.m. Panel F: Tumor volume quantificationof PDTX model final time point. Whiskers identify the maximum andminimum values; boxes indicate the 75^(th) and 25^(th) percentile andthe line the median. ** P<0.01 using two-tailed unpaired Student'st-test. Panel G: Waterfall plot showing tumor percent change frombaseline for PDTX model. Panel H: Representative images of PDTX tumors.Scale bar, 1 cm. Panel I: Representative H&E staining and IHC forphospho-histone H3 (PH3) and cleaved caspase-3 (CC3). Scale bars, 50 μm.Quantification of PH3- and CC3-positive foci (right panel). * P<0.05;**** P<0.0001 using one-way ANOVA. Data are represented as mean±s.e.m.

FIGS. 20A-20D, provide data demonstrating that CLCF1 and CNTFR areexpressed in non-small cell lung cancer (NSCLC).

DETAILED DESCRIPTION

Provided are agents that specifically bind a ligand of ciliaryneurotrophic factor receptor (CNTFR). In certain aspects, an agent ofthe present disclosure is a soluble CNTFR polypeptide. The soluble CNTFRpolypeptide may have an altered (e.g., reduced) binding affinity for oneor more ligand-CNTFR complex subunits, an altered (e.g., increased)binding affinity for one or more CNTFR ligands, or any combinationthereof. Compositions that include the agents of the present disclosureare also provided, as are methods of using the agents (e.g., fortreating a cell proliferative disorder) and methods of identifying anindividual as having a cell proliferative disorder associated with CNTFRsignaling.

Before the agents, compositions and methods of the present disclosureare described in greater detail, it is to be understood that the agents,compositions and methods are not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the agents, compositions and methods will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the agents, compositions andmethods. The upper and lower limits of these smaller ranges mayindependently be included in the smaller ranges and are also encompassedwithin the agents, compositions and methods, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the agents, compositions and methods.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the agents, compositions and methods belong. Althoughany agents, compositions and methods similar or equivalent to thosedescribed herein can also be used in the practice or testing of theagents, compositions and methods, representative illustrative agents,compositions and methods are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the materials and/or methods in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present agents, compositions and methods are notentitled to antedate such publication, as the date of publicationprovided may be different from the actual publication date which mayneed to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the agents, compositions andmethods, which are, for clarity, described in the context of separateembodiments, may also be provided in combination in a single embodiment.Conversely, various features of the agents, compositions and methods,which are, for brevity, described in the context of a single embodiment,may also be provided separately or in any suitable sub-combination. Allcombinations of the embodiments are specifically embraced by the presentdisclosure and are disclosed herein just as if each and everycombination was individually and explicitly disclosed, to the extentthat such combinations embrace operable processes and/or compositions.In addition, all sub-combinations listed in the embodiments describingsuch variables are also specifically embraced by the present agents,compositions and methods and are disclosed herein just as if each andevery such sub-combination was individually and explicitly disclosedherein.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentmethods. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

CNTFR Ligand-Binding Agents

As summarized above, aspects of the present disclosure include agentsthat specifically bind a ligand of ciliary neurotrophic factor receptor(CNTFR). CNTFR (also referred to as CNTF receptor subunit a) is a memberof the type 1 cytokine receptor family. CNTFR is the ligand-specificcomponent of a tripartite receptor for ciliary neurotrophic factor(CNTF), as well as other ligands such as cardiotrophin-like cytokinefactor 1 (CLCF1) and neuropoetin (NP). Binding of ligand to CNTFRrecruits the transmembrane components of the receptor, gp130 andleukemia inhibitory factor receptor (LIFR), facilitating signaltransduction.

As used herein, an “agent that specifically binds a ligand of ciliaryneurotrophic factor receptor (CNTFR)” is an agent that exhibits abinding affinity to one or more CNTFR ligands (e.g., one or more ofCLCF1, CNTFR, and/or NP) with a K_(D) of less than or equal to about10⁻⁵ M, less than or equal to about 10⁻⁶ M, less than or equal to about10⁻⁷ M, less than or equal to about 10⁻⁸ M, or less than or equal toabout 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M or less. Such affinities maybe readily determined using conventional techniques, such as byequilibrium dialysis; by using surface plasmon resonance (SPR)technology (e.g., the BIAcore 2000 instrument, using general proceduresoutlined by the manufacturer); by radioimmunoassay; or by another methodset forth in the examples below or known to the skilled artisan.

The agents of the present disclosure may be any suitable type of agentthat specifically binds a CNTFR ligand, including but not limited to,aptamers, antibodies, and the like. In certain aspects, the agent is asoluble CNTFR polypeptide.

Non-limiting embodiments of CNTFR ligand-binding agents of the presentdisclosure will now be described in detail.

Soluble CNTFR Polypeptides

As summarized above, in certain aspects, the agent that specificallybinds a ligand of CNTFR is a soluble CNTFR polypeptide. By “solubleCNTFR polypeptide” is meant a CNTFR polypeptide that is not integratedinto a cell membrane. The wild-type human CNTFR amino acid sequence(UniProtKB-P26992) is provided in Table 1 below.

TABLE 1 Wild-Type Human CNTFR Amino Acid Sequence (Non-Soluble)Amino Acid Sequence Wild-Type MAAPVPWACCAVLAAAAAVVYAQRHSPQEAPHVQHuman CNTFR YERLGSDVTLPCGTANWDAAVTWRVNGTDLAPDL (non-soluble)LNGSQLVLHGLELGHSGLYACFHRDSWHLRHQVL (SEQ ID NO: 1)LHVGLPPREPVLSCRSNTYPKGFYCSWHLPTPTY IPNTFNVTVLHGSKIMVCEKDPALKNRCHIRYMHLFSTIKYKVSISVSNALGHNATAITFDEFTIVKP DPPENVVARPVPSNPRRLEVTWQTPSTWPDPESFPLKFFLRYRPLILDQWQHVELSDGTAHTITDAYA GKEYIIQVAAKDNEIGTWSDWSVAAHATPWTEEPRHLTTEAQAAETTTSTTSSLAPPPTTKICDPGEL GSGGGPSAPFLVSVPITLALAAAAATASSLLI

According to certain embodiments, the soluble CNTFR polypeptide is notintegrated into a cell membrane by virtue of the polypeptide having oneor more solubility-conferring mutations. As used throughout the presentdisclosure, a “mutation” or “mutations” may include one or more aminoacid substitutions, one or more amino acid deletions (e.g.,truncations), one or more amino acid insertions, or any combinationthereof, in the relevant polypeptide, e.g., a CNTFR polypeptide of thepresent disclosure.

The one or more solubility-conferring mutations may be located in anysuitable region(s) of the CNTFR polypeptide. In certain aspects, thesoluble CNTFR polypeptide includes one or more solubility-conferringmutations in the domain that anchors wild-type CNTFR to the cellmembrane. This domain contains a lipidation site (S342) that ispost-translationally modified with glycosylphosphatidylinositol (GPI),which anchors the protein to the cell membrane. The wild-type humanCNTFR domain that anchors CNTFR to the cell membrane can be defined asconsisting of amino acids 343-372 as set forth in SEQ ID NO:1(underlined in Table 1). Under certain conditions, this portion of CNTFRis enzymatically modified to release CNTFR from the cell membrane.According to some embodiments, a soluble CNTFR polypeptide of thepresent disclosure includes a substitution mutation at S342 thatprecludes post-translational modification with GPI, thereby conferringsolubility. Wild-type human CNTFR also includes a signal peptideconsisting of amino acids 1-22 of SEQ ID NO:1 (underlined in Table 1).

According to certain embodiments, the CNTFR domain that anchors CNTFR tothe cell membrane includes one or more amino acid substitutions thatresult in the CNTFR polypeptide losing its ability to be anchored to acell membrane, thereby conferring solubility. Alternatively, oradditionally, the soluble CNTFR polypeptide may include a truncation(e.g., in the CNTFR domain that anchors CNTFR to the cell membrane) thatresults in the CNTFR polypeptide losing its ability to be anchored to acell membrane, thereby conferring solubility. In certain aspects, thesoluble CNTFR polypeptide lacks the CNTFR domain that anchors CNTFR tothe cell membrane. For example, the soluble CNTFR polypeptide may lackamino acids 343-372 set forth in SEQ ID NO:1.

In addition to optionally including one or more solubility-conferringmutations, a soluble CNTFR polypeptide of the present disclosure mayinclude one or more mutations that confer one or more other desirableproperties upon the polypeptide. Other desirable properties of interestinclude, but are not limited to, altered (e.g., greater) bindingaffinity for a CNTFR ligand, altered (e.g., greater) specificity for aparticular CNTFR ligand of interest as compared to one or more otherCNTFR ligands, altered (e.g., reduced) binding affinity for aligand-CNTFR complex subunit (e.g., gp130, LIFR, and/or the like),relative to a wild-type CNTF receptor, e.g., a receptor having the aminoacid sequence set forth in SEQ ID NO:1 or a mature form thereof.

By “greater binding affinity” or “increased binding affinity” is meantthat the soluble CNTFR polypeptide exhibits tighter binding (asindicated by a lower K_(D) value) to a molecule (e.g., a CNTFR ligandsuch as CLCF1) as compared to a wild-type CNTF receptor. By “lowerbinding affinity” or “reduced binding affinity” is meant that thesoluble CNTFR polypeptide exhibits less tight binding (as indicated by ahigher K_(D) value) to a molecule (e.g., a ligand-CNTFR complex subunitsuch as LIFR, gp130, or both) as compared to a wild-type CNTF receptor.

Methods are available for measuring the binding affinity of a CNTFRligand-binding agent (e.g., a soluble CNTFR polypeptide) to a moleculeof interest, e.g., a CNTFR ligand, a ligand-CNTFR complex subunit suchas LIFR, gp130, or the like. For example, surface plasmon resonance(SPR) technology (e.g., using a BIAcore™ 2000 instrument), KinExA®kinetic exclusion assay (Sapidyne Instruments), Bio-Layer Interferometry(BLI) technology (e.g., ForteBio Octet®), or other similarassay/technology may be employed to determine whether a CNTFRligand-binding agent exhibits a desired binding affinity. Suitableapproaches for measuring binding affinity in the context of the presentdisclosure include, e.g., those described in Hunter, S. A. and Cochran,J. R. (2016) Methods Enzymol. 580:21-44.

In some embodiments, in a direct binding assay, an equilibrium bindingconstant (K_(D)) may be measured using a CNTFR polypeptide conjugated toa fluorophore or radioisotope, or a CNTFR polypeptide that contains anN- or C-terminal epitope tag for detection by a labeled antibody. Iflabels or tags are not feasible or desired, a competition binding assaycan be used to determine the half-maximal inhibitory concentration(IC₅), the amount of unlabeled CNTFR polypeptide at which 50% of themaximal signal of the labeled competitor is detectable. A K_(D) valuecan then be calculated from the measured IC₅₀ value.

As summarized above, in certain aspects, a soluble CNTFR polypeptide ofthe present disclosure includes one or more mutations that alters (e.g.,reduces) the binding affinity of the soluble CNTFR polypeptide for aligand-CNTFR complex subunit relative to a wild-type CNTF receptor,e.g., a receptor having the amino acid sequence set forth in SEQ ID NO:1or a mature form thereof. By “ligand-CNTFR complex subunit” is meant aprotein that associates with wild-type CNTFR upon binding of CNTFR toligand. Non-limiting examples of ligand-CNTFR complex subunits includeleukemia inhibitory factor receptor (LIFR) and glycoprotein 130 (gp130).In certain aspects, the one or more mutations reduces the bindingaffinity of the soluble CNTFR polypeptide for LIFR, gp130, or both. Suchone or more mutations may prevent the soluble CNTFR polypeptide fromacting as an agonist upon binding to ligand, e.g., when it is desirableto reduce CNTFR-mediated signaling (e.g., to reduce cell proliferation).

According to certain embodiments, when the soluble CNTFR polypeptideexhibits reduced binding affinity for a ligand-CNTFR complex subunit,the binding affinity of the soluble CNTFR polypeptide has a K_(D) valuethat is 100 nM or greater in the presence of 10 nM of CLCF1.

In certain aspects, a soluble CNTFR polypeptide of the presentdisclosure has reduced binding affinity for LIFR and includes a mutation(e.g., an amino acid substitution) at amino acid position 177, 178, orboth, relative to a CNTFR polypeptide having the amino acid sequence setforth in SEQ ID NO:1. An example mutation at position 177 is Y177H.Another example mutation at position 177 is Y177A. An example mutationat position 178 is K178N. Another example mutation at position 178 isK178A. As demonstrated in the Examples section below, the presentinventors have determined that such mutations result in the solubleCNTFR polypeptide being an inhibitor of CNTFR signaling, whereas asoluble CNTFR polypeptide having unaltered affinity for ligand-CNTFRcomplex subunits acts as an agonist by virtue of its ability to recruit,e.g., LIFR and gp130 upon binding ligand. In certain aspects, a solubleCNTFR polypeptide of the present disclosure includes the mutations Y177Hand K178N, or the mutations Y177A and K178A, or the mutations Y177H andK178A, or the mutations Y177A and K178N.

According to certain embodiments, a soluble CNTFR polypeptide of thepresent disclosure has reduced binding affinity for gp130 and includes amutation (e.g., an amino acid substitution) at amino acid position 268,269, or both, relative to a CNTFR polypeptide having the amino acidsequence set forth in SEQ ID NO:1. An example mutation at position 268is T268A. An example mutation at position 269 is D269A. In certainaspects, a soluble CNTFR polypeptide of the present disclosure includesthe mutations T268A and D269A.

As summarized above, a soluble CNTFR polypeptide of the presentdisclosure may include one or more mutations that alters (e.g.,increases) the binding affinity and/or specificity of the soluble CNTFRpolypeptide for a CNTFR ligand of interest (e.g., CLCF1, NP, CNTF, oranother CNTFR ligand of interest) relative to a wild-type CNTF receptor,e.g., a receptor having the amino acid sequence set forth in SEQ ID NO:1or a mature form thereof. According to certain embodiments, when thesoluble CNTFR polypeptide exhibits increased binding affinity for aCNTFR ligand, the binding affinity of the soluble CNTFR polypeptide forthe ligand has a K_(D) value that is 10 nM or less.

According to certain embodiments, a soluble CNTFR polypeptide of thepresent disclosure includes one or more mutations that increases bindingaffinity and/or specificity for CLCF1. In certain aspects, such asoluble CNTFR polypeptide includes a mutation (e.g., an amino acidsubstitution) at amino acid position 110, 174, 237, 287, or anycombination thereof, relative to a CNTFR polypeptide having the aminoacid sequence set forth in SEQ ID NO:1. An example mutation at position110 is R110Q. An example mutation at position 174 is T174P. An examplemutation at position 237 is S237F. Another example mutation at position237 is S237Y. An example mutation at position 287 is I287F. In certainaspects, a soluble CNTFR polypeptide of the present disclosure includesone or any combination (e.g., each) of the mutations R110Q, T174P,S237F/S237Y, and I287F.

In some embodiments, a soluble CNTFR polypeptide of the presentdisclosure includes a mutation (e.g., an amino acid substitution) atamino acid position 110, 174, 177, 178, 237, 268, 269, 287, or anycombination thereof, relative to a CNTFR polypeptide having the aminoacid sequence set forth in SEQ ID NO:1.

In certain aspects, a soluble CNTFR polypeptide of the presentdisclosure includes one or any combination (e.g., each) of the mutationsR110Q, T174P, Y177H/Y177A, K178N/K178A, S237F/S237Y, T268A, D269A, andI287F.

A soluble CNTFR polypeptide according to one embodiment of the presentdisclosure includes the amino acid sequence set forth in Table 2 below(SEQ ID NO:2). In Table 2, mutations are bold/underlined. In thisexample, the soluble CNTFR polypeptide includes a C-terminal truncationof amino acids 343-372 relative to a wild-type CNTF receptor having theamino acid sequence set forth in SEQ ID NO:1. In certain aspects, such asoluble CNTFR polypeptide does not include a signal peptide (underlinedin Table 2).

TABLE 2 Amino Acid Sequence of an Example Soluble CNTFR PolypeptideAmino Acid Sequence Example MAAPVPWACCAVLAAAAAVVYAQRHSPQEAPHVQSoluble CNTFR YERLGSDVTLPCGTANWDAAVTWRVNGTDLAPDL PolypeptideLNGSQLVLHGLELGHSGLYACFHRDSWHLRHQVL SEQ ID NO: 2) LHVGLPP

EPVLSCRSNTYPKGFYCSWHLPTPTY (R110Q, T174P,IPNTFNVTVLHGSKIMVCEKDPALKNRCHIRYMH Y177H, K178N, LFS

IK

VSISVSNALGHNATAITFDEFTIVKP S237F, T268A,DPPENVVARPVPSNPRRLEVTWQTPSTWPDPE

F D269A, I287F) PLKFFLRYRPLILDQWQHVELSDGTAHTI

AYA GKEYIIQVAAKDNE

GTWSDWSVAAHATPWTEEP RHLTTEAQAAETTTSTTSSLAPPPTTKICDPGEL GS

Soluble CNTFR polypeptides of the present disclosure include (orcorrespond to) any of the CNTFR polypeptides presented in theExperimental section below and any of FIG. 7, FIG. 8 and FIG. 9.

According to certain embodiments, a soluble CNTFR polypeptide of thepresent disclosure includes an amino acid sequence that has 70% orgreater, 75% or greater, 80% or greater, 85% or greater, 90% or greater,95% or greater, or 99% or greater identity to amino acids 23-342 of SEQID NO:1 or SEQ ID NO:2, or a fragment thereof, such as a fragment havinga length of from 250 to 319 amino acids, 250 to 260 amino acids, 260 to270 amino acids, 270 to 280 amino acids, 280 to 290 amino acids, 290 to300 amino acids, 300 to 310 amino acids, or 310 to 319 amino acids. Inaddition to being soluble, such a CNTFR polypeptide may include one ormore desirable features, such as reduced binding affinity for one ormore ligand-CNTFR complex subunits (e.g., LIFR, gp130, or both),increased binding affinity/specificity for a CNTFR ligand (e.g., CLCF1),reduced binding affinity for a CNTFR ligand (e.g., CNTF, NP, etc.), andany combination thereof.

Antibodies

In certain aspects, the agent that specifically binds a ligand of CNTFRis an antibody. The terms “antibody”, “antibodies” and “immunoglobulin”include antibodies or immunoglobulins of any isotype, whole antibodies(e.g., antibodies composed of a tetramer which in turn is composed oftwo dimers of a heavy and light chain polypeptide); single chainantibodies; fragments of antibodies (e.g., fragments of whole or singlechain antibodies) which retain specific binding to the CNTFR ligand(e.g., CLCF1, NP and/or CNTF), including, but not limited to Fab, Fab′,Fv, scFv, and diabodies; chimeric antibodies; and humanized antibodies,e.g., humanized whole antibodies or humanized antibody fragments).

The “Fab” fragment contains the constant domain of the light chain andthe first constant domain (CH₁) of the heavy chain. Fab fragments differfrom Fab′ fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH₁ domain including one or more cysteinesfrom the antibody hinge region. Fab′-SH is the designation herein forFab′ in which the cysteine residue(s) of the constant domains bear afree thiol group. F(ab′)₂ antibody fragments originally were produced aspairs of Fab′ fragments which have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of an antibody, where these domains are present in asingle polypeptide chain. In some embodiments, the Fv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains, which enables the sFv to form the desired structure for antigenbinding.

In certain aspects, an antibody that specifically binds a ligand ofCNTFR specifically binds to CLCF1 preferentially as compared to CNTF orNP1. According to certain embodiments, an antibody that specificallybinds a ligand of CNTFR specifically binds to CLCF1 and not CNTF or NP1.

Engineering/Development and Production of CNTFR Ligand-Binding Agents

Also provided by the present disclosure are methods ofengineering/developing additional CNTFR ligand-binding agents having oneor more desired functionalities. The manner in which the CNTFRligand-binding agents are developed may vary. Rational and combinatorialapproaches may be used to engineer CNTFR ligand-binding agents withnovel properties, e.g., reduced binding affinity for one or moreligand-CNTFR complex subunits (e.g., LIFR, gp130, or both), increasedbinding affinity and/or specificity for a CNTFR ligand (e.g., CLCF1),reduced binding affinity for a CNTFR ligand (e.g., CNTF, NP, etc.), andany combination thereof. For example, to develop a soluble CNTFRpolypeptide or antibody, a library of CNTFR polypeptides or antibodiesmay be created and screened, e.g., by bacterial display, phage display,yeast surface display, fluorescence-activated cell sorting (FACS),and/or any other suitable screening method.

Yeast surface display is a powerful combinatorial technology that hasbeen used to engineer proteins with novel molecular recognitionproperties, increased target binding affinity, proper folding, andimproved stability. In this platform, libraries of protein variants aregenerated and screened in a high-throughput manner to isolate mutantswith desired biochemical and biophysical properties. As demonstrated inthe Examples section below, the present inventors have successfullyemployed yeast surface display for engineering CNTFR polypeptides withaltered binding affinities for CLCF1, LIFR and gp130 in a desirablemanner. Yeast surface display benefits from quality control mechanismsof the eukaryotic secretory pathway, chaperone-assisted folding, andefficient disulfide bond formation.

One example approach for developing a soluble CNTFR polypeptide having adesirable property of interest involves genetically fusing a CNTFRpolypeptide to the yeast mating agglutinin protein Aga2p, which isattached by two disulfide binds to the yeast cell wall protein Aga1p.This Aga2p-fusion construct, and a chromosomally integrated Aga1pexpression cassette, may be expressed under the control of a suitablepromoter, such as a galactose-inducible promoter. N- or C-terminalepitope tags may be included to measure cell surface expression levelsby flow cytometry using fluorescently labeled primary or secondaryantibodies. This construct represents the most widely used displayformat, where the N-terminus of the CNTFR polypeptide (or other proteinto be engineered) is fused to Aga2, but several alternative variationsof the yeast surface display plasmid have been described and may beemployed to develop a soluble CNTFR polypeptide of the presentdisclosure. One of the benefits of this screening platform overpanning-based methods used with phage or mRNA display is that two-colorFACS can be used to quantitatively discriminate clones that differ by aslittle as two-fold in binding affinity to a particular target.

To selectively mutate CNTFR at the DNA level, an example approach iserror prone PCR, which can be used to introduce mutations by any numberof altered reaction conditions including using a polymerase that doesnot possess proofreading (i.e. exonuclease) activity, using mixtures oftriphosphate derivatives of nucleoside analogues, using altered ratiosof dNTPs, varying concentrations of magnesium or manganese, or the like.Alternatively, degenerate codons can be introduced by oligonucleotideassembly using, e.g., overlap extension PCR. Next, the genetic materialmay be amplified using flanking primers with sufficient overlap with theyeast display vector for homologous recombination in yeast. Thesemethods allow CNTFR libraries to be created at relatively low cost andeffort. Synthetic libraries and recent methods have been developed thatallow defined control over library composition.

In certain aspects, a display library (e.g., a yeast display library) isscreened for binding to the target of interest (e.g., CNTFR ligand ofinterest, such as CLCF1) by FACS. Two-color FACS may be used for libraryscreening, where one fluorescent label can be used to detect the c-mycepitope tag and the other to measure interaction of the CNTFRpolypeptide against the binding target of interest. Different instrumentlasers and/or filter sets can be used to measure excitation and emissionproperties of the two fluorophores at single-cell resolution. Thisenables yeast expression levels to be normalized with binding. That is,a CNTFR polypeptide that exhibits poor yeast expression but binds a highamount of a target can be distinguished from a CNTFR polypeptide that isexpressed at high levels but binds weakly to a target. Accordingly, atwo-dimensional flow cytometry plot of expression versus binding willresult in a diagonal population of yeast cells that bind to targetantigen. High-affinity binders can be isolated using library sort gates.Alternatively, in an initial sort round it could be useful to clear thelibrary of undesired clones that do not express full-length protein.

Following enrichment of CNTFR libraries for clones encoding CNTFRpolypeptides of interest, the yeast plasmids are recovered andsequenced. Additional rounds of FACS can be performed under increasedsorting stringency. The binding affinities or kinetic off-rates ofindividual yeast-displayed CNTFR clones may then be measured.

Once CNTFR polypeptides of interest have been identified by surfacedisplay (e.g., yeast surface display), the engineered CNTFR polypeptidesmay be produced using a suitable method. According to certainembodiments, the CNTFR polypeptide is produced by solid phase peptidesynthesis. CNTFR polypeptide sequences may be synthesized using solidphase peptide chemistry on an automated synthesizer. For example,standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid phase peptidechemistry may be employed. Solid phase synthesis may be followed bypurification, e.g., by reversed-phase high-performance liquidchromatography (RP-HPLC).

In certain aspects, soluble CNTFR polypeptides are produced using arecombinant DNA approach. Strategies have been developed for producingproteins such as CNTFR polypeptides using recombinant methods in avariety of host cell types. For example, functional soluble CNTFRpolypeptides may be produced with barnase as a genetic fusion partner,which promotes folding in the E. coli periplasmic space and serves as auseful purification handle. According to certain embodiments, theengineered soluble CNTFR polypeptide is expressed in yeast (e.g., theyeast strain Pichia pastoris or Saccharomyces cerevesiae) or mammaliancells (e.g. human embryonic kidney cells or Chinese hamster ovarycells). The expression construct may encode one or more tags (e.g., aC-terminal hexahistadine tag for purification by, e.g., metal chelatingchromatography (Ni-NTA)). Size exclusion chromatography may then be usedto remove aggregates, misfolded multimers, and the like.

Aspects of the present disclosure include nucleic acids that encode theCNTFR ligand binding agents of the present disclosure. That is, providedare nucleic acids that encode any of the CNTFR ligand binding agentsdescribed herein (e.g., any of the soluble CNTFR polypeptides,antibodies, etc. described herein). In certain aspects, such a nucleicacid is present in an expression vector. The expression vector includesa promoter operably linked to the nucleic acid encoding the agent (e.g.,soluble CNTFR polypeptide), the promoter being selected based on thetype of host cell selected to express the agent. Suitable expressionvectors are typically replicable in the host organisms either asepisomes or as an integral part of the host chromosomal DNA. Commonly,expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance,kanamycin resistance, neomycin resistance, and/or the like) to permitdetection of those cells transformed with the desired DNA sequences.

Also provided are host cells that include a nucleic acid that encodesany of the CNTFR ligand binding agents described herein (e.g., any ofthe soluble CNTFR polypeptides, antibodies, etc. described herein), aswell as any expression vectors including the same. Escherichia coli isan example of a prokaryotic host cell that can be used for cloning anucleic acid encoding a CNTFR ligand binding agent of the presentdisclosure. Other microbial hosts suitable for use include bacilli, suchas Bacillus subtilis, and other enterobacteriaceae, such as Salmonella,Serratia, and various Pseudomonas species. In these prokaryotic hosts,one can also make expression vectors, which will typically containexpression control sequences compatible with the host cell (e.g., anorigin of replication). In addition, any number of a variety ofwell-known promoters will be present, such as the lactose promotersystem, a tryptophan (trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. The promoters willtypically control expression, optionally with an operator sequence, andhave ribosome binding site sequences and the like, for initiating andcompleting transcription and translation.

Other microbes, such as yeast, are also useful for expression.Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitableyeast host cells, with suitable vectors having expression controlsequences (e.g., promoters), an origin of replication, terminationsequences and the like as desired. Typical promoters include3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeastpromoters include, among others, promoters from alcohol dehydrogenase,isocytochrome C, and enzymes responsible for maltose and galactoseutilization.

In addition to microorganisms, mammalian cells (e.g., mammalian cellsgrown in in vitro cell culture) can also be used to express and producethe CNTFR ligand binding agents of the present disclosure. Suitablemammalian host cells include human cell lines, non-human primate celllines, rodent (e.g., mouse, rat) cell lines, and the like. Suitablemammalian cell lines include, but are not limited to, HeLa cells (e.g.,American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g.,ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573),Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHKcells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells,COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No.CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2cells, and the like. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter, and an enhancer, and necessary processing information sites,such as ribosome binding sites, RNA splice sites, polyadenylation sites,and transcriptional terminator sequences. Examples of suitableexpression control sequences are promoters derived from immunoglobulingenes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and thelike.

Once synthesized (either chemically or recombinantly), the CNTFR ligandbinding agents can be purified according to standard procedures known inthe art, including ammonium sulfate precipitation, affinity columns,column chromatography, high performance liquid chromatography (HPLC)purification, gel electrophoresis, and the like. A subject CNTFR ligandbinding agent can be substantially pure, e.g., at least about 80% to 85%pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or98% to 99%, or more, pure, e.g., free from contaminants such as celldebris, macromolecules other than the CNTFR ligand binding agent, etc.

Fusion Proteins and Conjugates

In certain aspects, provided are CNTFR ligand-binding agents (e.g., anyof the soluble CNTFR polypeptides or antibodies described herein) stablyassociated with (e.g., fused, conjugated, or otherwise attached to) aheterologous moiety.

In some embodiments, provided are fusion proteins in which a polypeptideCNTFR ligand-binding agent (e.g., any of the soluble CNTFR polypeptidesor antibodies of the present disclosure) is fused to a heterologouspolypeptide. Heterologous polypeptides of interest include, but are notlimited to, an Fc domain (e.g., a human or mouse Fc domain), an albumin,a transferrin, XTEN, a homo-amino acid polymer, a proline-alanine-serinepolymer, an elastin-like peptide, or any combination thereof. In certainaspects, the heterologous polypeptide increases the stability and/orserum half-life of the CNTFR ligand-binding agent upon itsadministration to an individual in need thereof, as compared to the sameCNTFR ligand-binding agent which is not fused to the heterologouspolypeptide. In certain aspects, provided are fusion proteins thatinclude any of the soluble CNTFR polypeptides of the present disclosurefused to a human Fc domain (e.g., a full-length human Fc domain orfragment thereof). According to certain embodiments, such a fusionprotein finds use, e.g., in administering to an individual in needthereof in accordance with the methods of the present disclosure (e.g.,an individual having a cell proliferative disorder associated with CNTFRsignaling). A non-limiting example of a human Fc domain that may befused to any of the soluble CNTFR polypeptides of the present disclosureis a human IgG1 Fc domain having the sequence set forth in Table 3 below(SEQ ID NO:3), or a fragment thereof.

TABLE 3 Amino Acid Sequence of an Example Human Fc DomainAmino Acid Sequence Example DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTHuman Fc CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY DomainRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ IDGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE NO: 3)WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

According to certain embodiments, provided are conjugates in which aCNTFR ligand-binding agent of the present disclosure is conjugated to amoiety. Moieties of interest include, but are not limited to,polyethylene glycol (PEG), an anti-cancer drug, a detectable label, andcombinations thereof.

Anti-cancer drugs of interest include agents that inhibit cellproliferation and/or kill cancer cells. Such agents may vary and includecytostatic agents and cytotoxic agents (e.g., an agent capable ofkilling a target cell tissue with or without being internalized into atarget cell). In certain aspects, the therapeutic agent is a cytotoxicagent selected from an enediyne, a lexitropsin, a duocarmycin, a taxane,a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid. In someembodiments, the cytotoxic agent is paclitaxel, docetaxel, CC-1065,CPT-11 (SN-38), topotecan, doxorubicin, morpholino-doxorubicin,rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin,combretastatin, calicheamicin, maytansine, maytansine DM1, maytansineDM4, DM-1, an auristatin or other dolastatin derivatives, such asauristatin E or auristatin F, AEB (AEB-071), AEVB (5-benzoylvalericacid-AE ester), AEFP (antibody-endostatin fusion protein), MMAE(monomethylauristatin E), MMAF (monomethylauristatin F),pyrrolobenzodiazepines (PBDs), eleutherobin, netropsin, or anycombination thereof. According to certain embodiments, the agent is aprotein toxin selected from hemiasterlin and hemiasterlin analogs suchas HTI-286 (e.g., see U.S. Pat. No. 7,579,323; WO 2004/026293; and U.S.Pat. No. 8,129,407, the full disclosures of which are incorporatedherein by reference), abrin, brucine, cicutoxin, diphtheria toxin,batrachotoxin, botulism toxin, shiga toxin, endotoxin, Pseudomonasexotoxin, Pseudomonas endotoxin, tetanus toxin, pertussis toxin, anthraxtoxin, cholera toxin, falcarinol, fumonisin BI, fumonisin B2, aflatoxin, maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin,scyllatoxin, hefutoxin, calciseptine, taicatoxin, calcicludine,geldanamycin, gelonin, lotaustralin, ocratoxin A, patulin, ricin,strychnine, trichothecene, zearlenone, and tetradotoxin. Enzymaticallyactive toxins and fragments thereof which may be employed includediphtheria A chain, non-binding active fragments of diphtheria toxin,exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes.

Detectable labels include labels that may be detected in an applicationof interest (e.g., in vitro and/or in vivo research and/or clinicalapplications). Detectable labels of interest include radioisotopes,enzymes that generate a detectable product (e.g., horseradishperoxidase, alkaline phosphatase, etc.), fluorescent proteins,paramagnetic atoms, and the like. In certain aspects, the CNTFRligand-binding agent is conjugated to a specific binding partner ofdetectable label (e.g., conjugated to biotin such that detection mayoccur via a detectable label that includes avidin/streptavidin).

According to certain embodiments, the agent is a labeling agent thatfinds use in in vivo imaging, such as near-infrared (NIR) opticalimaging, single-photon emission computed tomography (SPECT)/CT imaging,positron emission tomography (PET), nuclear magnetic resonance (NMR)spectroscopy, or the like. Labeling agents that find use in suchapplications include, but are not limited to, fluorescent labels,radioisotopes, and the like. In certain aspects, the labeling agent is amulti-modal in vivo imaging agent that permits in vivo imaging using twoor more imaging approaches (e.g., see Thorp-Greenwood and Coogan (2011)Dalton Trans. 40:6129-6143).

In certain aspects, the labeling agent is an in vivo imaging agent thatfinds use in near-infrared (NIR) imaging applications, which agent isselected from a Kodak X-SIGHT dye, Pz 247, DyLight 750 and 800 Fluors,Cy 5.5 and 7 Fluors, Alexa Fluor 680 and 750 Dyes, IRDye 680 and 800CWFluors. According to certain embodiments, the labeling agent is an invivo imaging agent that finds use in SPECT imaging applications, whichagent is selected from ^(99m)Tc, ¹¹¹In, ¹²³In, ²⁰¹Tl, and ¹³³Xe. Incertain aspects, the labeling agent is an in vivo imaging agent thatfinds use in positron emission tomography (PET) imaging applications,which agent is selected from ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁶⁴Cu, ⁶²Cu, ¹²⁴, ⁷⁶Br,⁸²Rb and ⁶⁸Ga.

Linkers that find use in the conjugates of the present disclosureinclude ester linkers, amide linkers, maleimide or maleimide-basedlinkers; valine-citrulline linkers; hydrazone linkers;N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linkers;Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)linkers; vinylsulfone-based linkers; linkers that include polyethyleneglycol (PEG), such as, but not limited to tetraethylene glycol; linkersthat include propanoic acid; linkers that include caproleic acid, andlinkers including any combination thereof.

Numerous strategies are available for linking a CNTFR ligand-bindingagent to a moiety of interest through a linker. For example, the moietyof interest may be derivatized by covalently attaching the linker to thedrug, where the linker has a functional group capable of reacting with a“chemical handle” on the CNTFR ligand-binding agent. The functionalgroup on the linker may vary and may be selected based on compatibilitywith the chemical handle on the CNTFR ligand-binding agent. According toone embodiment, the chemical handle on the CNTFR ligand-binding agent isprovided by incorporation of an unnatural amino acid having the chemicalhandle into the CNTFR ligand-binding agent. Such an unnatural amino acidmay be incorporated into a CNTFR ligand-binding agent, e.g., viachemical synthesis or recombinant approaches (e.g., using a suitableorthogonal amino acyl tRNA synthetase-tRNA pair for incorporation of theunnatural amino acid during translation in a host cell).

The functional group of an unnatural amino acid present in the CNTFRligand-binding agent may be an azide, alkyne, alkene, amino-oxy,hydrazine, aldehyde, nitrone, nitrile oxide, cyclopropene, norbornene,iso-cyanide, aryl halide, boronic acid, or other suitable functionalgroup, and the functional group on the linker is selected to react withthe functional group of the unnatural amino acid (or vice versa).

Compositions

Also provided are compositions that include a CNTFR ligand-binding agentof the present disclosure. The compositions may include any of the CNTFRligand-binding agents described herein, including any of the solubleCNTFR polypeptides and antibodies described herein.

In certain aspects, the compositions include a CNTFR ligand-bindingagent of the present disclosure present in a liquid medium. The liquidmedium may be an aqueous liquid medium, such as water, a bufferedsolution, and the like. One or more additives such as a salt (e.g.,NaCl, MgCl₂, KCl, MgSO₄), a buffering agent (a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), aprotease inhibitor, glycerol, and the like may be present in suchcompositions.

Pharmaceutical compositions are also provided. The pharmaceuticalcompositions include any of the CNTFR ligand-binding agents of thepresent disclosure (e.g., any of the soluble CNTFR polypeptides andantibodies of the present disclosure), and a pharmaceutically-acceptablecarrier. The pharmaceutical compositions generally include atherapeutically effective amount of the CNTFR ligand-binding agent. By“therapeutically effective amount” is meant a dosage sufficient toproduce a desired result, e.g., an amount sufficient to effectbeneficial or desired therapeutic (including preventative) results, suchas a reduction in cellular proliferation in an individual having a cellproliferative disorder associated with CNTFR signaling.

A CNTFR ligand-binding agent of the present disclosure can beincorporated into a variety of formulations for therapeuticadministration. More particularly, the CNTFR ligand-binding agent can beformulated into pharmaceutical compositions by combination withappropriate pharmaceutically acceptable excipients or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, injections, inhalants and aerosols.

Formulations of the CNTFR ligand-binding agents of the presentdisclosure suitable for administration to an individual (e.g., suitablefor human administration) are generally sterile and may further be freeof detectable pyrogens or other contaminants contraindicated foradministration to an individual according to a selected route ofadministration.

In pharmaceutical dosage forms, the CNTFR ligand-binding agent can beadministered alone or in appropriate association, as well as incombination, with other pharmaceutically-active compounds. The followingmethods and excipients are merely examples and are in no way limiting.

For oral preparations, the CNTFR ligand-binding agents can be used aloneor in combination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The CNTFR ligand-binding agents can be formulated into preparations forinjection by dissolving, suspending or emulsifying them in an aqueous ornon-aqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

The pharmaceutical composition may be in a liquid form, a lyophilizedform or a liquid form reconstituted from a lyophilized form, where thelyophilized preparation is to be reconstituted with a sterile solutionprior to administration. The standard procedure for reconstituting alyophilized composition is to add back a volume of pure water (typicallyequivalent to the volume removed during lyophilization); howeversolutions comprising antibacterial agents may be used for the productionof pharmaceutical compositions for parenteral administration.

An aqueous formulation of the CNTFR ligand-binding agent may be preparedin a pH-buffered solution, e.g., at pH ranging from about 4.0 to about7.0, or from about 5.0 to about 6.0, or alternatively about 5.5.Examples of buffers that are suitable for a pH within this range includephosphate-, histidine-, citrate-, succinate-, acetate-buffers and otherorganic acid buffers. The buffer concentration can be from about 1 mM toabout 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on thebuffer and the desired tonicity of the formulation.

Methods of Use

Also provided are methods of using the CNTFR ligand-binding agents andcompositions of the present disclosure. According to certainembodiments, provided are methods that include administering to anindividual in need thereof a therapeutically effective amount of a CNTFRligand-binding agent or pharmaceutical composition of the presentdisclosure, where binding of the agent to the ligand inhibits binding ofthe ligand to CNTFR. In certain aspects, the individual in need thereofhas a cell proliferative disorder associated with CNTFR signaling, andthe administering is effective in treating the cell proliferativedisorder. In certain aspects, the cell proliferative disorder is cancer.

For example, in some embodiments, a CNTFR ligand-binding agent orpharmaceutical composition of the present disclosure inhibits growth,metastasis and/or invasiveness of a cancer cell(s) in a host when theCNTFR ligand-binding agent or pharmaceutical composition is administeredin an effective amount. By “cancer cell” is meant a cell exhibiting aneoplastic cellular phenotype, which may be characterized by one or moreof, for example, abnormal cell growth, abnormal cellular proliferation,loss of density dependent growth inhibition, anchorage-independentgrowth potential, ability to promote tumor growth and/or development inan immunocompromised non-human animal model, and/or any appropriateindicator of cellular transformation. “Cancer cell” may be usedinterchangeably herein with “tumor cell”, “malignant cell” or “cancerouscell”, and encompasses cancer cells of a solid tumor, a semi-solidtumor, a primary tumor, a metastatic tumor, and the like.

Cancers which may be treated using the methods of the present disclosureinclude, but are not limited to, solid tumors, lung cancer (e.g.,non-small cell lung cancer (NSCLC), breast cancer, prostate cancer,pancreatic cancer, colorectal carcinoma, renal cell carcinoma, Hodgkin'slymphoma, Non-Hodgkin's lymphoma, anaplastic large cell lymphoma, acutemyelogenous leukemia, multiple myeloma, and any other type of cancerwhich may be treated using a CNTFR ligand-binding agent orpharmaceutical composition of the present disclosure.

The CNTFR ligand-binding agent may be administered alone (e.g., inmonotherapy) or in combination (e.g., in combination therapy) with oneor more additional therapeutic agents.

In some embodiments, an effective amount of the CNTFR ligand-bindingagent (or pharmaceutical composition including same) is an amount that,when administered alone (e.g., in monotherapy) or in combination (e.g.,in combination therapy) with one or more additional therapeutic agents,in one or more doses, is effective to reduce the symptoms of a cellproliferative disorder (e.g., cancer) in the individual by at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, or more, compared to the symptoms in the individual inthe absence of treatment with the CNTFR ligand-binding agent orpharmaceutical composition.

In certain aspects, the methods of the present disclosure inhibitgrowth, metastasis and/or invasiveness of cancer cells in the individualwhen the CNTFR ligand-binding agent or pharmaceutical composition isadministered in an effective amount.

The CNTFR ligand-binding agent or pharmaceutical composition may beadministered to an individual using any available method and routesuitable for drug delivery, including in vivo and ex vivo methods, aswell as systemic and localized routes of administration. Conventionaland pharmaceutically acceptable routes of administration includeintranasal, intramuscular, intra-tracheal, subcutaneous, intradermal,topical application, ocular, intravenous, intra-arterial, nasal, oral,and other enteral and parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe CNTFR ligand-binding agent and/or the desired effect. The CNTFRligand-binding agents or pharmaceutical compositions may be administeredin a single dose or in multiple doses. In some embodiments, the CNTFRligand-binding agent or pharmaceutical composition is administeredintravenously. In some embodiments, the CNTFR ligand-binding agent orpharmaceutical composition is administered by injection, e.g., forsystemic delivery (e.g., intravenous infusion) or to a local site.

A variety of individuals are treatable according to the subject methods.Generally such subjects are “mammals” or “mammalian,” where these termsare used broadly to describe organisms which are within the classmammalia, including the orders carnivore (e.g., dogs and cats), rodentia(e.g., mice, guinea pigs, and rats), and primates (e.g., humans,chimpanzees, and monkeys). In some embodiments, the individual will behuman.

By “treating” or “treatment” is meant at least an amelioration of thesymptoms associated with the cell proliferative disorder (e.g., cancer)of the individual, where amelioration is used in a broad sense to referto at least a reduction in the magnitude of a parameter, e.g. symptom,associated with the cell proliferative disorder being treated. As such,treatment also includes situations where the cell proliferativedisorder, or at least symptoms associated therewith, are completelyinhibited, e.g., prevented from happening, or stopped, e.g., terminated,such that the individual no longer suffers from the cell proliferativedisorder, or at least the symptoms that characterize the cellproliferative disorder.

Dosing is dependent on severity and responsiveness of the disease stateto be treated. Optimal dosing schedules can be calculated frommeasurements of drug accumulation in the body of the patient. Theadministering physician can determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual CNTFR ligand-binding agents, andcan generally be estimated based on EC₅₀s found to be effective in invitro and in vivo animal models, etc. In general, dosage is from 0.01 μgto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly. The treating physician can estimaterepetition rates for dosing based on measured residence times andconcentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the subject undergomaintenance therapy to prevent the recurrence of the disease state,where the CNTFR ligand-binding agent or pharmaceutical composition isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every several months, onceevery six months, once every year, or at any other suitable frequency.

The therapeutic methods of the present disclosure may includeadministering a single type of CNTFR ligand-binding agent to a subject,or may include administering two or more types of CNTFR ligand-bindingagents to a subject by administration of a cocktail of different CNTFRligand-binding agents, where the CNTFR ligand-binding agents areengineered to bind to, e.g., distinct CNTFR ligands.

In some embodiments, the methods include, prior to the administering theCNTFR ligand-binding agent or pharmaceutical composition, identifyingthe individual as having a cell proliferative disorder associated withCNTFR signaling. Identifying the individual as having a cellproliferative disorder associated with CNTFR signaling may be carriedout using a variety of approaches and combinations thereof. In certainaspects, the identifying is based on CNTFR ligand abundance in a sampleobtained from the individual. The CNTFR ligand may be one or more ofCNTF, CLCF1, NP, etc., and any combinations thereof. In certain aspects,the sample includes cancer-associated fibroblasts (CAFs, such as normallung fibroblasts (NLFs)), and identifying the individual as having acell proliferative disorder associated with CNTFR signaling is based onthe level of CLCF1 expression in the CAFs. In some embodiments, theCNTFR ligand abundance is quantified using a soluble CNTFR polypeptideas a CNTFR ligand capture agent, e.g., any of the soluble CNTFRpolypeptides of the present disclosure. For example, the soluble CNTFRpolypeptide used as a CNTFR ligand capture agent may be any of thesoluble CNTFR polypeptides described herein.

According to certain embodiments, the identifying is based on CNTFRabundance and/or the abundance of CNTFR-gp130-LIFR tripartite receptorcomplexes in a sample obtained from the individual. In certain aspects,the identifying is based on the level of CNTFR signaling in a sampleobtained from the individual. The level of CNTFR signaling may be basedon the phosphorylation status of one or more CNTFR signaling pathwaymolecules. For example, the present inventors have determined thatbinding of CNTFR to CLCF1 results in activation of the Jak-STAT andRas-Raf-MEK-ERK signaling pathways. As such, the abundance, activity,phosphorylation status, and/or the like of any of Jak, STAT, Ras, Raf,MEK, ERK, or any combination thereof, may be assessed to determineaberrant CNTFR signaling in the individual.

The identifying may be based on ligands, CNTFR molecules,CNTFR-gp130-LIFR tripartite receptor complexes, etc. quantified usingany suitable approaches. According to certain embodiments, theidentifying is based on an immunoassay. A variety of suitableimmunoassay formats are available, including ELISA, flow cytometryassays, immunohistochemistry on tissue section samples,immunofluorescence on tissue section samples, Western analysis, and/orthe like.

In some embodiments, the identifying is based on nucleic acidsequencing. For example, the number of sequencing reads corresponding toan mRNA encoding a protein of interest may be used to determine theexpression level of the protein. In certain aspects, the sequencing isperformed using a next-generation sequencing system, such as on asequencing platform provided by Illumina® (e.g., the HiSeq™, MiSeq™and/or Genome Analyzer™ sequencing systems); Ion Torren™ (e.g., the IonPGM™ and/or Ion Proton™ sequencing systems); Pacific Biosciences (e.g.,the PACBIO RS II sequencing system); Life Technologies™ (e.g., a SOLIDsequencing system); Roche (e.g., the 454 GS FLX+ and/or GS Juniorsequencing systems); or any other sequencing platform of interest.Protocols for isolating nucleic acids from tissue or fluid samples, aswell as protocols for preparing sequencing libraries having sequencingadapters appropriate for the desired sequencing platform are readilyavailable.

In some embodiments, methods that include identifying the individual ashaving a cell proliferative disorder associated with CNTFR signalingfurther include obtaining the sample from the individual.

Also provided are methods that include identifying an individual ashaving a cell proliferative disorder associated with CNTFR signaling.Identifying the individual as having a cell proliferative disorderassociated with CNTFR signaling may be carried out using a variety ofapproaches and combinations thereof. In certain aspects, the identifyingis based on CNTFR ligand abundance in a sample obtained from theindividual. The CNTFR ligand may be one or more of CNTF, CLCF1, NP,etc., and any combinations thereof. In certain aspects, the sampleincludes cancer-associated fibroblasts (CAFs, such as normal lungfibroblasts (NLFs)), and identifying the individual as having a cellproliferative disorder associated with CNTFR signaling is based on thelevel of CLCF1 expression in the CAFs. In some embodiments, the CNTFRligand abundance is quantified using a soluble CNTFR polypeptide as aCNTFR ligand capture agent, e.g., any of the soluble CNTFR polypeptidesof the present disclosure. For example, the soluble CNTFR polypeptideused as a CNTFR ligand capture agent may be any of the soluble CNTFRpolypeptides described herein. According to certain embodiments, theidentifying is based on CNTFR abundance and/or the abundance ofCNTFR-gp130-LIFR tripartite receptor complexes in a sample obtained fromthe individual. In certain aspects, the identifying is based on thelevel of CNTFR signaling in a sample obtained from the individual. Thelevel of CNTFR signaling may be based on the phosphorylation status ofone or more CNTFR signaling pathway molecules. For example, the presentinventors have determined that binding of CNTFR to CLCF1 results inactivation of the Jak-STAT and Ras-Raf-MEK-ERK signaling pathways. Assuch, the abundance, activity, phosphorylation status, and/or the likeof any of Jak, STAT, Ras, Raf, MEK, ERK, or any combination thereof, maybe assessed to determine aberrant CNTFR signaling in the individual. Theidentifying may be based on ligands, CNTFR molecules, CNTFR-gp130-LIFRtripartite receptor complexes, etc. quantified using any suitableapproaches, such as by immunoassay, nucleic acid sequencing, and/or thelike. In some embodiments, the methods further include obtaining thesample from the individual.

The sample obtained from the individual may be any sample suitable fordetermining whether the individual has a cell proliferative disorderassociated with CNTFR signaling. In certain aspects, the sample is afluid sample, such as whole blood, serum, plasma, or the like. In someembodiments, the sample is a tissue sample. Tissue samples of interestinclude, but are not limited to, tumor biopsy samples, and the like.

Kits

Also provided by the present disclosure are kits. According to certainembodiments, the kits include a therapeutically effective amount of anyof the CNTFR ligand-binding agents described herein, or any of thepharmaceutical compositions described herein, and instructions foradministering the CNTFR ligand-binding agent or pharmaceuticalcomposition to an individual in need thereof (e.g., an individualidentified as having a cell proliferative disorder associated with CNTFRsignaling. In certain aspects, the kits include a CNTFR ligand-bindingagent or a pharmaceutical composition of the present disclosure, presentin a container. The container may be a tube, vial, or the like.According to certain embodiments, the kit includes the CNTFRligand-binding agent or the pharmaceutical composition present in one ormore unit dosages, such as 1, 2 or more, 3 or more, 4 or more, 5 ormore, etc. unit dosages.

Components of the kits may be present in separate containers, ormultiple components may be present in a single container.

The instructions for administering the CNTFR ligand-binding agent orpharmaceutical composition to an individual may be recorded on asuitable recording medium. For example, the instructions may be printedon a substrate, such as paper or plastic, etc. As such, the instructionsmay be present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with thepackaging or sub-packaging) etc. In other embodiments, the instructionsare present as an electronic storage data file present on a suitablecomputer readable storage medium, e.g., portable flash drive, DVD,CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, the means for obtaining theinstructions is recorded on a suitable substrate.

In some embodiments, provided are kits that include a CNTFR ligandcapture agent and instructions for using the capture agent to quantify aCNTFR ligand abundance present in a biological sample. The CNTFR ligandmay be one or more of CNTF, CLCF1, NP, etc., and any combinationsthereof. In some embodiments, the CNTFR ligand abundance is quantifiedusing a soluble CNTFR polypeptide as a CNTFR ligand capture agent, e.g.,any of the soluble CNTFR polypeptides of the present disclosure. Forexample, the soluble CNTFR polypeptide used as a CNTFR ligand captureagent may be any of the soluble CNTFR polypeptides described herein.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental Introduction

The communication between epithelial cells and their underlying stromais an important but poorly understood aspect of organismal biology. Ifaberrantly regulated, these interactions can prove to be tumorigenic.Although cancer-associated fibroblasts (CAFs) are known to promote andsustain the growth of tumors, the underlying mechanisms remainincompletely understood. The inventors have identified a novel mechanismof communication in which CAFs secrete cardiotrophin-like cytokinefactor 1 (CLCF1), a cytokine that binds ciliary neurotrophic factorreceptor (CNTFR) on tumor cells and promotes neoplastic growth.

It was hypothesized that a soluble CNTFR polypeptide could be employedas a therapeutic to reduce CNTFR signaling, and that a soluble CNTFRpolypeptide having a greater binding affinity for CLCF1 as compared towild-type CNTFR would be desirable. To engineer a soluble CNTFRpolypeptide having a greater binding affinity for CLCF1, mutations wererandomly introduced into CNTFR through error-prone PCR. The resultinglibrary was displayed as fusion proteins on the yeast cell surface(schematically illustrated in FIG. 4, panel A), where a subset retainedbinding to CLCF1.

Example 1—Recombinant Expression of Functional CLCF1

CLCF1 was cloned into the bacterial expression plasmid pET28b andtransformed into Rosetta-gami 2(DE)3 cells. When expressed, recombinantCLCF1 accumulated in inclusion bodies, which were solubilized in 60%ddH2O, 40% acetonitrile, 0.1% TFA) containing 5 mM DTT. Reversed-phasehigh performance liquid chromatography was used to purify CLCF1.Treatment of the human non-small cell lung cancer cell line A549 withCLCF1 induced phosphorylation of STAT3 at Tyr705 within minutes (FIG. 2,panel A). Activation of STAT3 was also detected in two other cell linesH23 and H358 (FIG. 2, panel B). These results show that CLCF1 triggersan increase in phosphorylation of Tyr705 in STAT3.

Example 2—Recombinant CLCF1 Increases Cell Survival

Cytokine mediated STAT3 activation has been shown to protect cells fromapoptosis. To test whether CLCF1 can also increase cell survival, A549cells were treated with CLCF1 following 24 hours of serum starvation.After 72 hours of incubation, CLCF1 was shown to increase cell survivalin a concentration dependent manner (FIG. 3, panel A). When tested onH23 cells, again, higher CLCF1 concentration led to increased survival(FIG. 3, panel B). Such effect of CLCF1 treatment was not seen whenserum was included in the assay suggesting that the effect is moreapparent in stressor-induced conditions such as serum starvation.

Example 3—Yeast-Displayed CNTFR Binds to Recombinantly Expressed CLCF1

The extracellular domain of CNTFR was cloned into the yeast surfacedisplay plasmid pCTCON2 and transformed into the Saccharomycescerevisiae strain EBY 100. This plasmid enables expression of CNTFR onthe yeast cell surface through genetic linkage to the yeast agglutininprotein Aga2p (FIG. 4, panel A). CNTFR was flanked by N-terminalhemagglutinin (HA) and C-terminal c-Myc tags, which allows for detectionof full-length protein by antibody labeling and flow cytometrydetection. Induced yeast demonstrated an expression percentage of 50%,and the expressing population mostly bound recombinantly expressed CLCF1when incubated with 20 nM CLCF, as measured by flow cytometry using asecondary antibody (FIG. 4, panel B).

Example 4—Yeast-Displayed CNTFR Forms a Tripartite Receptor Complex withCLCF1. gp130. and LIFR

As mentioned previously, the CNTFR-CLCF1 complex binds to gp130 and LIFRto form a tripartite receptor complex. Therefore, a fully functionalCNTFR is expected to bind to the two beta subunits when incubated withCLCF1. To prepare soluble constructs of LIFR and gp130, theextracellular domains were cloned into mammalian expression plasmid Add2and transfected into human embryonic kidney (HEK) 293 suspension cells.The constructs were prepared as His-tag and mouse IgG2a fusions tofacilitate purification. The His-tagged constructs were purified usingnickel affinity chromatography, and the Fc-fusion constructs werepurified using protein A affinity chromatography. When yeast-displayedCNTFR was incubated with LIFR-Fc, no binding signal was detected, whichwas expected since without CLCF1, CNTFR does not bind to the betareceptors to activate the downstream signaling pathways (FIG. 5, panelsA and B). However, upon addition of CLCF1, LIFR-Fc binding signalingincreased, indicating that in the presence of CLCF1, CNTFR indeedinteracts with LIFR (FIG. 5, panel C). When yeast displayed-CNTFR wasincubated with CLCF1 and gp130, LIFR-Fc binding signal increased evenhigher, indicating that gp130 further contributes to binding betweenLIFR and CNTFR (FIG. 5, panels D and E).

Several previous studies suggest that CNTFR interaction with the betareceptors may occur in a specific order. For example, it has beenproposed that CNTF first binds to CNTFR, then recruits gp130, andfinally complex with LIFR. The above experiments show that binding ofCNTFR to LIFR can occur in the absence gp130 as long as CLCF1 bindsfirst. In addition, without CLCF1, gp130-Fc showed no detectable bindingto yeast-displayed CNTFR (FIG. 6, panels A and B). Upon addition ofCLCF1 gp130-Fc signal increased significantly, indicating that gp130 canbind to CNTFR without LIFR (FIG. 6, panel C). When LIFR was added, therewas a slight increase of gp130 binding signal. Together, the set ofbinding experiments shown in FIG. 6 suggests that there is synergisticor additive binding between LIFR and gp130 to CNTFR-CLCF1 (FIG. 6,panels D and E).

Example 5—Affinity Maturation of CNTFR to CLCF1 Using Yeast SurfaceDisplay

A protein engineering strategy was designed in which mutations wererandomly introduced into the extracellular domain of CNTFR (20-342)using error-prone PCR. The resulting library, with an estimateddiversity of about 1×10⁸ transformants, was displayed as fusion proteinson the yeast cell surface as previously described. Using equilibriumbinding conditions and screened by flow cytometric sorting (FACS), thelibrary was enriched for variants showing increased CLCF1 binding,normalized for a given amount of c-Myc expression. Screening stringencywas imparted by decreasing the concentration of CLCF1 incubated with thelibrary in each successive round of sorting. After 3 rounds of screeningusing FACS, three yeast populations with different levels of CLCF1binding signals were observed. Consensus mutations appeared in the twohighest CLCF1 binding populations: T174P was observed in theintermediate affinity population, while S237F was observed in thehighest affinity population (FIG. 7 and FIG. 8).

Example 6—Screening a Shuffled CNTFR Library to Identify High AffinityCLCF1 Variants

Despite the enriched consensus mutations, the sorted library stillcontained substantial diversity. To test additive effects of themutations found in the two higher binding populations, the clones wereshuffled using Staggered Extension Process (StEP) method. To imposeincreased stringency with this library, a combination of equilibriumbinding and kinetic off-rate screens were used. After 3 rounds ofscreening, a combination of four mutations (R110Q, T174P, S237F, andI287F) emerged (FIG. 9). Qualitative yeast-displayed binding studiessuggested that each of these mutations contributed to the bindingaffinity for CLCF1, and thus the clone (variant 4) containing all fourmutations was chosen for further experiments (FIG. 10, panels A and B).Interestingly, while yeast displayed wild-type CNTFR bound to CNTF,variant 4 did not bind to CNTF indicating that the process of directedevolution led to increased specificity for CLCF1 (FIG. 10, panel C).Variant 4 still bound to mCLCF1 in addition to hCLCF1 (FIG. 10, panelD).

Example 7—Negative Screening of Affinity Matured CNTFR for ReducedBinding to LIFR

Alanine substitutions at CNTFR amino acid residues 268 and 269 werepreviously shown to decrease CNTFR interaction with the beta receptors.When T268A and D269A were introduced into CNTFR displayed on the yeastcell surface, we discovered that these mutations reduced binding forgp130 but significant binding to LIFR remained (FIG. 11). Thus, tofurther engineer CNTFR to decrease its binding for LIFR, another randommutation using error-prone PCR introduced into variant 4 from FIG. 10,panel B. The resulting library was again displayed on yeast, but thistime screened for the population that had decreased binding signal forLIFR in the presence of CLCF1. To retain the binding affinity for CLCF1,the screening was performed by alternating between positive selectionfor CLCF1 binding and negative selection for LIFR (FIG. 12). After sixrounds of sorting, two consensus mutation emerged (Y177H and K178N)(FIG. 13, panel A). Because these mutations additively contributed todecreased LIFR binding a clone with both mutations (named eCNTFR) waschosen for further characterization. eCNTFR contains the four mutationsthat confer high affinity CLCF1 binding from variant 4, as well as thetwo new mutations that decrease binding to LIFR.

Example 8—Characterization of Soluble Engineered CNTFR

The eCNTFR variant was cloned into the Add2 mammalian expression vectorand transfected into HEK293 cells to produce soluble constructs fused toHis-tag and mouse IgG2a Fc. While wild-type CNTFR (wtCNTFR) yielded 8milligrams per liter of cell culture, eCNTFR yielded 4 milligrams perliter. To measure the binding affinity of the soluble CNTFR constructs,they were first incubated with CLCF1 at room temperature for 4 hours.Then the complexes were captured using microtiter plates coated withanti-his antibody or anti-mouse Fc antibody. Using a rabbit anti-CLCF1primary antibody, followed by an HRP tagged anti-rabbit secondaryantibody, a binding interaction between CLCF1 and soluble CNTFR was ableto be measured. Both His tagged and Fc fusion eCNTFR showed picomolarbinding affinity to CLCF1 (FIG. 14, panel B). In comparison, the CLCF1binding affinity was too weak to be quantified for wtCNTFR. The sameapproach was used to characterize binding interaction with the betareceptors. In these experiments, eCNTFR-His and eCNTFR-Fc showed nodetectable binding signal to gp130 and LIFR, unlike wtCNTFR which boundboth receptors (FIG. 13, panel B). These results were consistent withthe results observed from the yeast-displayed constructs. Becauseincreasing the size of a small protein to avoid glomerular filtrationcan significantly increase serum half-life, we chose eCNTFR-Fc, fused tomouse IgG2a Fc domain, for evaluating downstream effects of CLCF1inhibition. This Fc fusion would also benefit from FcRn-mediatedrecycling for half-life extension in vivo.

The amino acid sequence of the example soluble CNTFR polypeptide-Fcfusion (“eCNTFR-Fc”—SEQ ID NO:4) is set forth in Table 4 below. In thisexample, the soluble CNTFR polypeptide of SEQ ID NO:2 is fused to afull-length mouse Fc region (underlined in Table 4).

TABLE 4 Amino Acid Sequence of an Example SolubleCNTFR Polypeptide-Fc Fusion Amino Acid Sequence ExampleMAAPVPWACCAVLAAAAAVVYAQRHSPQEAPHVQ Soluble CNTFRYERLGSDVTLPCGTANWDAAVTWRVNGTDLAPDL PolypeptideLNGSQLVLHGLELGHSGLYACFHRDSWHLRHQVL (SEQ ID NO: 4)LHVGLPPQEPVLSCRSNTYPKGFYCSWHLPTPTY (R110Q, T174P,IPNTFNVTVLHGSKIMVCEKDPALKNRCHIRYMH Y177H, K178N,LFSPIKHNVSISVSNALGHNATAITFDEFTIVKP S237F, T268A,DPPENVVARPVPSNPRRLEVTWQTPSTWPDPEFF D269A, I287F)PLKFFLRYRPLILDQWQHVELSDGTAHTIAAAYA GKEYIIQVAAKDNEFGTWSDWSVAAHATPWTEEPRHLTTEAQAAETTTSTTSSLAPPPTTKICDPGEL GSRRLEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISW FVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRA PQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVE KKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

Example 9—eCNTFR-Fc Inhibits STAT3 Activation in Human NSCLC Cells

To determine whether eCNTFR-Fc could effectively neutralize CLCF1 andinhibit STAT3 phosphorylation we tested its effect on two human NSCLCcell lines, A549 and H23. The cells were stimulated with CLCF1 in thepresence and absence of the soluble CNTFR constructs. Strikingly,wtCNTFR-Fc increased phosphorylation of STAT3 (Tyr705) while treatmentwith eCNTFR-Fc effectively decreased the STAT3 phosphorylation (FIG. 15,panel A). Furthermore, incubation with eCNTFR-Fc inhibitedCLCF1-mediated cell survival in serum starved conditions in both A549and H23 cells (FIG. 15, panels B and C, respectively).

Example 10—eCNTFR-Fc Decreases Tumor Growth in Mouse Xenograft Models

Next, we tested whether eCNTFR-Fc retained its ability to sequesterCLCF1 and decrease tumor growth in vivo. Non-tumor bearing mice weregiven a single dose of eCNTFR-Fc at 1 mg/kg body weight and the amountof unbound CLCF1 in the serum at different time points was measured withELISA using eCNTFR-Fc as a capturing agent. Serum samples were collectedand analyzed 6, 12, 24, 36, and 48 hours after injection. Serum CLCF1was rapidly sequestered by injected eCNTFR-Fc, but after 48 h CLCF1levels approached the untreated baseline (FIG. 16, panel A). Startingfrom 6 h after injection the measured eCNTFR-Fc level decreased with theestimated half-life of around 48 h, which correlated with thesequestration of serum CLCF1.

To test whether eCNTFR-Fc could affect tumor growth in vivo, twodifferent models of non-small cell lung cancer were treated witheCNTFR-Fc. In the first model, A549 cells were subcutaneously injectedand grown on two opposing flanks of each mouse. After the tumor sizesgrew to approximately 100 mm³ the mice were injected with 10 mg/kg or 1mg/kg body weight or saline control, twice weekly for 4 weeks. Treatmentwith both concentrations of eCNTFR-Fc significantly decreased tumorgrowth after 17 days of treatment compared with saline control (FIG. 16,panels B and C).

Another experiment was conducted in a similar manner, using a xenograftmodel with H23 cells. This time, eCNTFR-Fc treatment at 10 mg/kg wascompared with wtCNTFR-Fc treatment and saline control, and injectionfrequency was increased to three times per week for 39 days. Similarresults were observed, where eCNTFR-Fc decreased the tumor volumesignificantly by the end of the study while wtCNTFR-Fc seemed toslightly increase the tumor size although these results werestatistically insignificant (FIG. 17).

Example 11—Specificity in Engineered CLCF1 Trap (eCNTFR)

CNTF (ciliary neurotrophic factor) is another ligand for CNTFR thatshares with CLCF1 the ligand binding motif of CNTFR. Since CNTF-mediatedsignaling is important for neuronal cell survival and may havecytoprotective effects induced by injury, binding affinity of eCNTFR-Fcto CLCF1 over CNTF can be favorable for preventing side effects frominhibiting CNTF. When the binding affinity of eCNTFR-Fc forrecombinantly produced CNTF was measured using ELISA, while wtCNTFR-Fcdemonstrated substantial binding for CNTF, eCNTFR-Fc lost its affinityfor CNTF in the tested concentrations. On the other hand, eCNTFR-Fcshowed high affinity for mouse CLCF1 (mCLCF1), which may be critical forin vivo mouse model experiments since mouse CLCF1 can phosphorylateSTAT3 in human cells. Data is shown in FIG. 18, panel A.

To determine whether eCNTFR-Fc could effectively neutralize CLCF1 andinhibit STAT3 phosphorylation, its effect was tested on two human NSCLCcell lines: A549 and H23. The cells were stimulated with CLCF1 in thepresence and absence of soluble CNTFR constructs. While wtCNTFR-Fcincreased phosphorylation of STAT3 (Tyr705), eCNTFR-Fc effectivelydecreased the phosphorylation in both cell lines. Data is shown in FIG.18, panel B.

Example 12—Pharmacokinetics of CLCF1 Sequestration and eCNTFR-FcClearance

To test whether eCNTFR-Fc retained its ability to sequester CLCF1 invivo, blood clearance and CLCF1 sequestration after intraperitoneal(i.p.) dosing of 10 mg/kg eCNTFR-Fc in NOD/SCID/gamma mice werequantified. Serum samples were collected post injection and unboundCLCF1 (squares) was measured by ELISA using eCNTFR-Fc as the capturingagent. Vehicle treated mice were used to determined baseline CLCF1levels. eCNTFR-Fc levels (circles) in the blood were quantified by ELISAusing an anti-Fc antibody as the capturing agent. Serum CLCF1 wasrapidly sequestered by injected eCNTFR-Fc, however, at 72 hours, CLCF1levels approached the untreated baseline. (FIG. 19, panel A). These datademonstrate that eCNTFR-Fc effectively binds to mouse CLCF1 and suggestthat any significant toxicities in vivo should be observable in mice.

Example 13—eCNTFR-Fc Inhibits In Vivo Tumor Growth

To test the pharmacological efficacy of eCNTFR in vivo, NSCLC cell lineswere engrafted in immunodeficient mice. Primary tumors were allowed togrow to an approximate size of 100-150 mm3. Initially, mice wererandomized into one of four groups: vehicle (PBS), wildtype CNTFR(wtCNTFR-Fc), a low dose (1 mg/kg) of eCNTFR-Fc, and a high dose (10mg/kg) of eCNTFR-Fc (FIG. 21, panel B). Mice were injected twice perweek by intraperitoneal (i.p.) injection for 18 days. Administration ofeCNTFR-Fc demonstrated dose-dependent tumor inhibition (FIG. 19, panelsB-D). Comparable effects were observed in a second NSCLC cell line.

Increasing evidence suggests that PDTXs faithfully recapitulate humantumor biology and predict responses to therapy. PDTXs obtained by directimplants of surgically resected tumors from humans into mice are knownto maintain morphological similarities and recapitulate molecularprofiles of the original tumors. As such, to validate our findings in aclinically relevant setting, we generated NSCLC PDTX models to testeCNTFR-Fc. One LUAD PDTX model showed significant tumor growthinhibition upon eCNTFR-Fc treatment three times per week by i.p.injection for three weeks or until control (PBS) mice showed signs ofmorbidity (FIG. 19, panels E-H).

In each xenograft model, proliferation and apoptosis in response toeCNTFR were assayed by PH3 and CC3 immunostaining, respectively. Asignificant decrease in proliferation and an increase in apoptosis wasobserved after treatment with eCNTFR-Fc (FIG. 19, panel 1). In the lowerleft and right bar graphs in panel 1, the results for PBS, WT CNTFR,eCNTFR (1 mg/kg), and eCNTFR (10 mg/kg), are provided from left toright. Taken together, these results indicate that disruption ofCLCF1-induced signaling in tumor cells can be effectively achieved usingeCNTFR.

Example 15—Expression of CLCF1 and CNTFR in Lung Cancer

While cancer-associated fibroblasts (CAFs) express CLCF1 and may be thesource for this cytokine in vivo, the present study determined thatNSCLC cell lines also secrete CLCF1, suggesting the existence of bothparacrine and autocrine signaling mechanisms for this cytokine (FIG. 20,panel A). The receptor for CLCF1, CNTFR, was also determined to beexpressed on all NSCLC cell lines and patient-derived xenograft (PDTX)models tested (FIG. 20, panels B and C). Expression of CNTFR was alsoobserved by immunohistochemistry in PDTX models and in tumors generatedin the KrasG12D;P53f/f genetically-engineered mouse model (FIG. 20,panel D). Taken together these results suggest that the CLCF1-CNTFRsignaling axis is active in lung adenocarcinoma and that it may have arole in oncogenesis, particularly in tumors driven by oncogenic Kras.

Notwithstanding the appended claims, the disclosure is also defined bythe following clauses:

1. A pharmaceutical composition, comprising:

an agent that specifically binds a ligand of ciliary neurotrophic factorreceptor (CNTFR); and

a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of Clause 1, wherein the agent thatspecifically binds a ligand of CNTFR specifically binds a ligandselected from the group consisting of: ciliary neurotrophic factor(CNTF), cardiotrophin-like cytokine factor 1 (CLCF1), neuropoetin (NP),and any combination thereof.3. The pharmaceutical composition of Clause 1 or Clause 2, wherein theagent that specifically binds a ligand of CNTFR is a soluble CNTFRpolypeptide.4. The pharmaceutical composition of Clause 3, wherein the soluble CNTFRpolypeptide comprises a mutation that reduces binding affinity of thesoluble CNTFR polypeptide for a ligand-CNTFR complex subunit relative toa wild-type CNTFR polypeptide.5. The pharmaceutical composition of Clause 4, wherein the ligand-CNTFRcomplex subunit is glycoprotein 130 (gp130), leukemia inhibitory factorreceptor (LIFR), or both.6. The pharmaceutical composition of Clause 5, wherein the ligand-CNTFRcomplex subunit is LIFR.7. The pharmaceutical composition of Clause 6, wherein the mutation thatreduces binding affinity for LIFR is at amino acid position 177, 178, orboth, relative to a CNTFR polypeptide having the amino acid sequence setforth in SEQ ID NO:1.8. The pharmaceutical composition of Clause 5, wherein the ligand-CNTFRcomplex subunit is gp130.9. The pharmaceutical composition of Clause 8, wherein the mutation thatreduces binding affinity for gp130 is at amino acid position 268, 269,or both, relative to a CNTFR polypeptide having the amino acid sequenceset forth in SEQ ID NO:1.10. The pharmaceutical composition of any one of Clauses 3 to 9, whereinthe soluble CNTFR polypeptide comprises a solubility-conferring mutationin the domain that anchors wild-type CNTFR to a cell membrane.11. The pharmaceutical composition of Clause 10, wherein the solubleCNTFR polypeptide comprises a truncation in the domain that anchorswild-type CNTFR to a cell membrane.12. The pharmaceutical composition of Clause 10, wherein the solubleCNTFR polypeptide lacks the domain that anchors wild-type CNTFR to acell membrane.13. The pharmaceutical composition of one of Clauses 3 to 12, whereinthe soluble CNTFR polypeptide comprises a mutation that increasesbinding affinity of the soluble CNTFR polypeptide for a CNTFR ligandrelative to a wild-type CNTFR polypeptide.14. The pharmaceutical composition of Clause 13, wherein the CNTFRligand is selected from the group consisting of: ciliary neurotrophicfactor (CNTF), cardiotrophin-like cytokine factor 1 (CLCF1), neuropoetin(NP), and any combination thereof.15. The pharmaceutical composition of Clause 13, wherein the CNTFRligand is CLCF1.16. The pharmaceutical composition of Clause 15, wherein the mutationthat increases binding affinity for CLCF1 is at amino acid position 110,174, 237, 287, or any combination thereof, relative to a CNTFRpolypeptide having the amino acid sequence set forth in SEQ ID NO:1.17. The pharmaceutical composition of Clause 1 or Clause 2, wherein theagent is an antibody.18. The pharmaceutical composition of any one of Clauses 1 to 17,wherein the agent is a polypeptide fused to a heterologous polypeptide.19. The pharmaceutical composition of Clause 18, wherein theheterologous polypeptide is an Fc domain, an albumin, a transferrin,XTEN, a homo-amino acid polymer, a proline-alanine-serine polymer, anelastin-like peptide, or any combination thereof.20. The pharmaceutical composition of Clause 19, wherein theheterologous polypeptide is an Fc domain.21. The pharmaceutical composition of Clause 20, wherein the Fc domainis a human Fc domain.22. The pharmaceutical composition of any one of Clauses 1 to 21,wherein the agent is conjugated to a moiety.23. The pharmaceutical composition of Clause 22, wherein the moiety ispolyethylene glycol (PEG), an anti-cancer drug, a detectable label, orany combination thereof.24. A method, comprising:

administering to an individual in need thereof a therapeuticallyeffective amount of the pharmaceutical composition of any one of Clauses1 to 23 or the soluble CNTFR polypeptide of any one of Clauses 58 to 74,wherein binding of the agent to the ligand inhibits binding of theligand to CNTFR.

25. The method according to Clause 24, wherein the individual in needthereof has a cell proliferative disorder associated with CNTFRsignaling, and the administering is effective in treating the cellproliferative disorder.26. The method according to Clause 24 or Clause 25, further comprising,prior to the administering, identifying the individual as having a cellproliferative disorder associated with CNTFR signaling.27. The method according to Clause 26, wherein the identifying is basedon CNTFR ligand abundance in a sample obtained from the individual.28. The method according to Clause 27, wherein the abundance is of aCNTFR ligand selected from the group consisting of: CNTF, CLCF1, NP, andany combination thereof.29. The method according to Clause 27 or Clause 28, wherein the CNTFRligand abundance is quantified using a soluble CNTFR polypeptide as aCNTFR ligand capture agent.30. The method according to Clause 29, wherein the soluble CNTFRpolypeptide used as a CNTFR ligand capture agent is the soluble CNTFRpolypeptide of any one of Clauses 58 to 74.31. The method according to Clause 26, wherein the identifying is basedon CNTFR abundance in a sample obtained from the individual.32. The method according to Clause 26, wherein the identifying is basedon the level of CNTFR signaling in a sample obtained from theindividual.33. The method according to Clause 32, wherein quantifying CNTFRsignaling in the sample comprises quantifying the phosphorylation statusof one or more CNTFR signaling pathway molecules.34. The method according to any one of Clauses 27 to 33, wherein theidentifying is based on an immunoassay.35. The method according to any one of Clauses 27 to 33, wherein theidentifying is based on nucleic acid sequencing.36. The method according to any one of Clauses 27 to 35, wherein thesample is a tissue sample.37. The method according to any one of Clauses 27 to 35, wherein thesample is a fluid sample.38. The method according to any one of Clauses 27 to 37, furthercomprising obtaining the sample from the individual.39. The method according to any one of Clauses 25 to 38, wherein thecell proliferative disorder is cancer.40. The method according to Clause 39, wherein the cancer is lungcancer.41. The method according to Clause 40, wherein the lung cancer isnon-small cell lung cancer (NSCLC).42. A method, comprising:

identifying an individual as having a cell proliferative disorderassociated with CNTFR signaling.

43. The method according to Clause 42, wherein the identifying is basedon CNTFR ligand abundance in a sample obtained from the individual.44. The method according to Clause 43, wherein the abundance is of aCNTFR ligand selected from the group consisting of: CNTF, CLCF1, NP, andany combination thereof.45. The method according to Clause 42 or Clause 43, wherein the CNTFRligand abundance is quantified using a soluble CNTFR polypeptide as aCNTFR ligand capture agent.46. The method according to Clause 45, wherein the soluble CNTFRpolypeptide used as a CNTFR ligand capture agent is the soluble CNTFRpolypeptide of any one of Clauses 58 to 74.47. The method according to Clause 42, wherein the identifying is basedon CNTFR abundance in a sample obtained from the individual.48. The method according to Clause 42, wherein the identifying is basedon the level of CNTFR signaling in a sample obtained from theindividual.49. The method according to Clause 48, wherein the level of CNTFRsignaling in the sample is determined based on the phosphorylationstatus of one or more CNTFR signaling pathway molecules.50. The method according to any one of Clauses 42 to 49, wherein theidentifying is based on an immunoassay.51. The method according to any one of Clauses 42 to 49, wherein theidentifying is based on nucleic acid sequencing.52. The method according to any one of Clauses 42 to 51, wherein thesample is a tissue sample.53. The method according to any one of Clauses 42 to 51, wherein thesample is a fluid sample.54. The method according to any one of Clauses 43 to 53, furthercomprising obtaining the sample from the individual.55. The method according to any one of Clauses 42 to 54, wherein thecell proliferative disorder is cancer.56. The method according to Clause 55, wherein the cancer is lungcancer.57. The method according to Clause 56, wherein the lung cancer isnon-small cell lung cancer (NSCLC).58. A soluble CNTFR polypeptide, comprising:

-   -   a mutation that reduces binding affinity of the soluble CNTFR        polypeptide for a ligand-CNTFR complex subunit relative to a        wild-type CNTFR polypeptide,    -   a mutation that increases binding affinity of the soluble CNTFR        polypeptide for a CNTFR ligand relative to a wild-type CNTFR        polypeptide, or    -   both.        59. The soluble CNTFR polypeptide of Clause 58, wherein the        soluble CNTFR polypeptide comprises a mutation that reduces        binding affinity of the soluble CNTFR polypeptide for a        ligand-CNTFR complex subunit relative to a wild-type CNTFR        polypeptide, and wherein the ligand-CNTFR complex subunit is        glycoprotein 130 (gp130), leukemia inhibitory factor receptor        (LIFR), or both.        60. The soluble CNTFR polypeptide of Clause 59, wherein the        ligand-CNTFR complex subunit is LIFR.        61. The soluble CNTFR polypeptide of Clause 60, wherein the        mutation that reduces binding affinity for LIFR is at amino acid        position 177, 178, or both, relative to a CNTFR polypeptide        having the amino acid sequence set forth in SEQ ID NO:1.        62. The soluble CNTFR polypeptide of Clause 59, wherein the        ligand-CNTFR complex subunit is gp130.        63. The soluble CNTFR polypeptide of Clause 62, wherein the        mutation that reduces binding affinity for gp130 is at amino        acid position 268, 269, or both, relative to a CNTFR polypeptide        having the amino acid sequence set forth in SEQ ID NO:1.        64. The soluble CNTFR polypeptide of any one of Clauses 58 to        63, wherein the soluble CNTFR polypeptide comprises a        solubility-conferring mutation in the domain that anchors        wild-type CNTFR to a cell membrane.        65. The soluble CNTFR polypeptide of Clause 64, wherein the        soluble CNTFR polypeptide comprises a truncation in the domain        that anchors wild-type CNTFR to a cell membrane.        66. The soluble CNTFR polypeptide of Clause 64, wherein the        soluble CNTFR polypeptide lacks the domain that anchors        wild-type CNTFR to a cell membrane.        67. The soluble CNTFR polypeptide of one of Clauses 58 to 66,        wherein the soluble CNTFR polypeptide comprises a mutation that        increases binding affinity of the soluble CNTFR polypeptide for        a CNTFR ligand relative to a wild-type CNTFR polypeptide.        68. The soluble CNTFR polypeptide of Clause 67, wherein the        CNTFR ligand is selected from the group consisting of: ciliary        neurotrophic factor (CNTF), cardiotrophin-like cytokine factor 1        (CLCF1), neuropoetin (NP), and any combination thereof.        69. The soluble CNTFR polypeptide of Clause 67, wherein the        CNTFR ligand is CLCF1.        70. The soluble CNTFR polypeptide of Clause 69, wherein the        mutation that increases binding affinity for CLCF1 is at amino        acid position 110, 174, 237, 287, or any combination thereof,        relative to a CNTFR polypeptide having the amino acid sequence        set forth in SEQ ID NO:1.        71. The soluble CNTFR polypeptide of one of Clauses 58 to 70,        wherein the soluble CNTFR polypeptide is fused to a heterologous        polypeptide.        72. The soluble CNTFR polypeptide of Clause 71, wherein the        heterologous polypeptide is an Fc domain, an albumin, a        transferrin, XTEN, a homo-amino acid polymer, a        proline-alanine-serine polymer, an elastin-like peptide, or any        combination thereof.        73. The soluble CNTFR polypeptide of Clause 71, wherein the        heterologous polypeptide is an Fc domain.        74. The soluble CNTFR polypeptide of Clause 73, wherein the Fc        domain is a human Fc domain.        75. A nucleic acid that encodes the soluble CNTFR polypeptide of        any one of Clauses 58 to 74.        76. An expression vector comprising the nucleic acid of Clause        75.        77. A host cell comprising the soluble CNTFR polypeptide of any        one of Clauses 58 to 70, the nucleic acid of Clause 75, the        expression vector of Clause 76, or any combination thereof.        78. The host cell of Clause 77, wherein the host cell is a        prokaryotic cell.        79. The host cell of Clause 77, wherein the host cell is a        eukaryotic cell.        80. The host cell of Clause 79, wherein the eukaryotic cell is a        mammalian cell.        81. The host cell of Clause 80, wherein the mammalian cell is a        human cell.        82. A nucleic acid that encodes a CNTFR polypeptide fused to a        cell surface display protein.        83. The nucleic acid of Clause 82, wherein the CNTFR polypeptide        comprises one or more mutations relative to a CNTFR polypeptide        having the amino acid sequence set forth in SEQ ID NO:1.        84. The nucleic acid of Clause 83, wherein the cell surface        display protein is selected from the group consisting of: a        bacterial surface display protein, a phage display protein, and        a yeast display protein.        85. The nucleic acid of Clause 84, wherein the cell surface        display protein is a yeast display protein.        86. The nucleic acid of Clause 85, wherein the yeast display        protein is Aga2p.        87. An expression vector comprising the nucleic acid of any one        of Clauses 82 to 86.        88. A host cell comprising the nucleic acid of any one of        Clauses 82 to 86, or the expression vector of Clause 87.        89. A CNTFR polypeptide encoded by the nucleic acid of any one        of Clauses 82 to 86, or the expression vector of Clause 87.

Accordingly, the preceding merely illustrates the principles of thepresent disclosure. It will be appreciated that those skilled in the artwill be able to devise various arrangements which, although notexplicitly described or shown herein, embody the principles of theinvention and are included within its spirit and scope. Furthermore, allexamples and conditional language recited herein are principallyintended to aid the reader in understanding the principles of theinvention and the concepts contributed by the inventors to furtheringthe art, and are to be construed as being without limitation to suchspecifically recited examples and conditions. Moreover, all statementsherein reciting principles, aspects, and embodiments of the invention aswell as specific examples thereof, are intended to encompass bothstructural and functional equivalents thereof. Additionally, it isintended that such equivalents include both currently known equivalentsand equivalents developed in the future, i.e., any elements developedthat perform the same function, regardless of structure. The scope ofthe present invention, therefore, is not intended to be limited to theexemplary embodiments shown and described herein. Rather, the scope andspirit of present invention is embodied by the appended claims.

1-89. (canceled)
 90. A method of treating a disorder associated withCNTFR signaling in an individual in need thereof, the method comprising:administering to the individual a therapeutically effective amount of asoluble ciliary neurotrophic factor receptor (CNTFR) polypeptidecomprising a CNTFR extracellular domain that specifically bindscardiotrophin-like cytokine factor 1 (CLCF1), wherein the extracellulardomain comprises: one or more mutations that reduce binding affinity ofthe soluble CNTFR polypeptide for leukemia inhibitory factor receptor(LIFR) relative to a wild-type CNTFR polypeptide, wherein the one ormore mutations comprise a mutation at amino acid position 177, 178, orboth, relative to a CNTFR polypeptide having the amino acid sequence setforth in SEQ ID NO:1; and/or one or more mutations that increase bindingaffinity of the soluble CNTFR polypeptide for CLCF1 relative to awild-type CNTFR polypeptide, wherein the one or more mutations comprisea mutation at amino acid position 110, 174, 237, 287, or any combinationthereof, relative to a CNTFR polypeptide having the amino acid sequenceset forth in SEQ ID NO:1.
 91. The method according to claim 90, whereinthe one or more mutations that reduce binding affinity of the solubleCNTFR polypeptide for LIFR comprise Y177H, K178N, or both.
 92. Themethod according to claim 90, wherein the one or more mutations thatincrease binding affinity of the soluble CNTFR polypeptide for CLCF1comprise R110Q, T174P, S237F, I287F, or any combination thereof.
 93. Themethod according to claim 90, wherein the soluble CNTFR polypeptidecomprises a mutation at each of amino acid positions 110, 174, 177, 178,237, and
 287. 94. The method according to claim 93, wherein the solubleCNTFR polypeptide comprises each of the following mutations: R110Q,T174P, Y177H, K178N, S237F, and I287F.
 95. The method according to claim90, wherein the soluble CNTFR polypeptide comprises one or moremutations that reduce binding affinity of the soluble CNTFR polypeptidefor glycoprotein 130 (gp130) relative to a wild-type CNTFR polypeptide.96. The method according to claim 95, wherein the one or more mutationsthat reduce binding affinity of the soluble CNTFR polypeptide for gp130comprise a mutation at amino acid position 268, 269, or both, relativeto a CNTFR polypeptide having the amino acid sequence set forth in SEQID NO:1.
 97. The method according to claim 96, wherein the one or moremutations that reduce binding affinity for gp130 comprise T268A, D269A,or both.
 98. The method according to claim 90, wherein the individual inneed thereof has a cell proliferative disorder associated with CNTFRsignaling, and the administering is effective in treating the cellproliferative disorder.
 99. The method according to claim 98, whereinthe cell proliferative disorder is cancer.
 100. The method according toclaim 99, wherein the cancer is lung cancer.
 101. The method accordingto claim 100, wherein the lung cancer is non-small cell lung cancer(NSCLC).
 102. A pharmaceutical composition comprising: a soluble CNTFRpolypeptide comprising a CNTFR extracellular domain that specificallybinds CLCF1, wherein the extracellular domain comprises: one or moremutations that reduce binding affinity of the soluble CNTFR polypeptidefor LIFR relative to a wild-type CNTFR polypeptide, wherein the one ormore mutations comprise a mutation at amino acid position 177, 178, orboth, relative to a CNTFR polypeptide having the amino acid sequence setforth in SEQ ID NO:1; and/or one or more mutations that increase bindingaffinity of the soluble CNTFR polypeptide for CLCF1 relative to awild-type CNTFR polypeptide, wherein the one or more mutations comprisea mutation at amino acid position 110, 174, 237, 287, or any combinationthereof, relative to a CNTFR polypeptide having the amino acid sequenceset forth in SEQ ID NO:1; and a pharmaceutically acceptable carrier.103. The pharmaceutical composition of claim 102, wherein the one ormore mutations that reduce binding affinity of the soluble CNTFRpolypeptide for LIFR comprise Y177H, K178N, or both.
 104. Thepharmaceutical composition of claim 102, wherein the one or moremutations that increase binding affinity of the soluble CNTFRpolypeptide for CLCF1 comprise R110Q, T174P, S237F, 1287F, or anycombination thereof.
 105. The pharmaceutical composition of claim 102,wherein the soluble CNTFR polypeptide comprises a mutation at each ofamino acid positions 110, 174, 177, 178, 237, and
 287. 106. Thepharmaceutical composition of claim 105, wherein the soluble CNTFRpolypeptide comprises each of the following mutations: R110Q, T174P,Y177H, K178N, S237F, and 1287F.
 107. The pharmaceutical composition ofclaim 102, wherein the soluble CNTFR polypeptide comprises one or moremutations that reduce binding affinity of the soluble CNTFR polypeptidefor glycoprotein 130 (gp130) relative to a wild-type CNTFR polypeptide.108. The pharmaceutical composition of claim 102, wherein the solubleCNTFR polypeptide comprises one or more mutations that reduce bindingaffinity of the soluble CNTFR polypeptide for glycoprotein 130 (gp130)relative to a wild-type CNTFR polypeptide.
 109. The pharmaceuticalcomposition of claim 108, wherein the one or more mutations that reducebinding affinity of the soluble CNTFR polypeptide for gp130 comprise amutation at amino acid position 268, 269, or both, relative to a CNTFRpolypeptide having the amino acid sequence set forth in SEQ ID NO:1.